Two mitofusin proteins, mammalian homologues of FZO, with distinct functions are both required for mitochondrial fusion

Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. Although these morphologic changes are considered crucial for cellular functions, the underlying mechanisms remain elusive, especially in mammalian cells. We characterized two rat mitochondrial outer membrane proteins, Mfn1 and Mfn2, with distinct tissue expressions, that are homologous to Drosophila Fzo, a GTPase involved in mitochondrial fusion. Expression of the GTPase-domain mutant of Mfn2 (Mfn2(K109T)) in HeLa cells induced mitochondrial fragmentation in which Mfn2(K109T) localized at the restricted domains. Immuno-electronmicroscopy revealed that Mfn2(K109T) was concentrated at the contact domains between adjacent mitochondria, suggesting that fusion of the outer membrane was arrested at some intermediate step. Mfn1 expression induced highly connected tubular network structures depending on the functional GTPase domain. The Mfn1-induced tubular networks were suppressed by co-expression with Mfn2. In vivo depletion of either isoform by RNA interference revealed that both are required to maintain normal mitochondrial morphology. The fusion of differentially-labeled mitochondria in HeLa cells subjected to depletion of either Mfn isoform and subsequent cell fusion by hemagglutinating virus of Japan revealed that both proteins have distinct functions in mitochondrial fusion. We conclude that the two Mfn isoforms cooperate in mitochondrial fusion in mammalian cells.     

Role of sodium in mitochondrial membrane depolarization induced by P2X7 receptor activation in submandibular glands

The effect of ATP on mitochondrial membrane depolarization in rat submandibular glands was investigated. Exposure of the cell suspension to high concentrations of ATP induced a sustained depolarization of mitochondrial membrane. This effect was blocked in the presence of magnesium and reproduced by low concentrations of 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP), suggesting the implication of the P2X(7) purinergic receptor. This point was confirmed by comparison of the response to ATP by wild-type and P2X(7) knock-out (P2X(7)R(-/-)) mice. Mitochondria took up calcium after ATP stimulation but the depolarization of the mitochondrial membrane by ATP was not affected by the removal of calcium from the extracellular medium. It was nearly fully suppressed in the absence of sodium and partially blocked by the mitochondrial Na/Ca exchanger inhibitor 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157). Both ATP and monensin increased the uptake of extracellular sodium (as shown by the depolarization of the plasma membrane) but the sodium ionophore did not affect the mitochondrial membrane potential. It is concluded that the activation of P2X(7) receptors depolarizes the mitochondrial membrane. The uptake of extracellular sodium is necessary but not sufficient to induce this response.     

Bz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax

Bz-423 is a proapoptotic 1,4-benzodiazepine with potent therapeutic properties in murine models of lupus and psoriasis. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. Herein, we report the signaling pathway activated by Bz-423 in mouse embryonic fibroblasts containing knockouts of key apoptotic proteins. Bz-423-induced superoxide activates cytosolic ASK1 and its release from thioredoxin. A mitogen-activated protein kinase cascade follows, leading to the specific phosphorylation of JNK. JNK signals activation of Bax and Bak which then induces mitochondrial outer membrane permeabilization to cause the release of cytochrome c and a commitment to apoptosis. The response of these cells to Bz-423 is critically dependent on both superoxide and JNK activation as antioxidants and the JNK inhibitor SP600125 prevents Bax translocation, cytochrome c release, and cell death. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a sequential and specific apoptotic response. Collectively, these data suggest that the selectivity of Bz-423 observed in vivo results from cell-type specific differences in redox balance and signaling by ASK1 and Bcl-2 proteins.     

Magainin 2 amide and analogues. Antimicrobial activity, membrane depolarization and susceptibility to proteolysis

We compared the abilities of synthetic magainin 2 amide and its analogues to inhibit the growth of Escherichia coli and to cause membrane depolarization in E. coli cells and cytochrome oxidase liposomes. The analogue, magainin A, was about 40-times more active than magainin 2 amide in inhibiting the growth of E. coli and had a much more sustained effect on the membrane potential. In the liposomal system, however, there was only approx. 20% difference between these two peptides in the reduction of membrane potential and uncoupling of respiration. Studies with pronase digestion suggested that the difference in potency may be due to differential susceptibility to proteolysis in the presence of membranes.     

Effects of cryopreservation on Ca2+ signals induced by membrane depolarization, caffeine, thapsigargin and progesterone in boar spermatozoa

Although the fertilizing ability of spermatozoa is greatly reduced after freezing, complete understanding of alterations induced by cryopreservation has not been elucidated. The present study evaluates the effects of cryopreservation on the Ca2+ handling of boar spermatozoa using several sperm activators. Intracellular Ca2+ signals from single spermatozoa were measured using confocal Ca2+ imaging of unfrozen samples and of other spermatozoa after having been frozen. Elevation of the external K+ concentration elicited a three times larger Ca2+ increase in fresh spermatozoa than in cryopreserved spermatozoa. Caffeine elicited Ca2+ transients with some oscillations in the fresh spermatozoa, but not in the thawed spermatozoa. Depletion of the Ca2+ store with thapsigargin induced a rapid rise in Ca2+ in the control but generated a smaller increase of Ca2+ after thawing. Exposure to progesterone induced a biphasic rise of the Ca2+ level in the fresh spermatozoa only. Sperm viability was reduced by cryopreservation. Resting Ca2+ levels in fresh and cryopreserved spermatozoa were similar. Longer incubation (2.5 h) of thawed spermatozoa partly recovered the Ca2+ response to the interventions. These results suggest that cryopreservation reduces the responsiveness of spermatozoa to depolarization, modulators of the internal Ca2+ store and progesterone in terms of the Ca2+ signal, thus providing a possible mechanism for reduced fertility observed in cryopreserved boar spermatozoa.     

Loss of caspase-9 reveals its essential role for caspase-2 activation and mitochondrial membrane depolarization

Caspase-9 plays an important role in apoptosis induced by genotoxic stress. Irradiation and anticancer drugs trigger mitochondrial outer membrane permeabilization, resulting in cytochrome c release and caspase-9 activation. Two highly contentious issues, however, remain: It is unclear whether the loss of the mitochondrial membrane potential DeltaPsi(M) contributes to cytochrome c release and whether caspases are involved. Moreover, an unresolved question is whether caspase-2 functions as an initiator in genotoxic stress-induced apoptosis. In the present study, we have identified a mutant Jurkat T-cell line that is deficient in caspase-9 and resistant to apoptosis. Anticancer drugs, however, could activate proapoptotic Bcl-2 proteins and cytochrome c release, similarly as in caspase-9-proficient cells. Interestingly, despite these alterations, the cells retained DeltaPsi(M). Furthermore, processing and enzyme activity of caspase-2 were not observed in the absence of caspase-9. Reconstitution of caspase-9 expression restored not only apoptosis but also the loss of DeltaPsi(M) and caspase-2 activity. Thus, we provide genetic evidence that caspase-9 is indispensable for drug-induced apoptosis in cancer cells. Moreover, loss of DeltaPsi(M) can be functionally separated from cytochrome c release. Caspase-9 is not only required for DeltaPsi(M) loss but also for caspase-2 activation, suggesting that these two events are downstream of the apoptosome.     

MARCH-V is a novel mitofusin 2- and Drp1-binding protein able to change mitochondrial morphology

Mitofusins and Drp1 are key components in mitochondrial membrane fusion and division, but the molecular mechanism underlying the regulation of their activities remains to be clarified. Here, we identified human membrane-associated RING-CH (MARCH)-V as a novel transmembrane protein of the mitochondrial outer membrane. Immunoprecipitation studies demonstrated that MARCH-V interacts with mitofusin 2 (MFN2) and ubiquitinated forms of Drp1. Overexpression of MARCH-V promoted the formation of long tubular mitochondria in a manner that depends on MFN2 activity. By contrast, mutations in the RING finger caused fragmentation of mitochondria. We also show that MARCH-V promotes ubiquitination of Drp1. These results indicate that MARCH-V has a crucial role in the control of mitochondrial morphology by regulating MFN2 and Drp1 activities.     

Cell fusion,haemolysis and mitochondrial swelling induced by retinol and derivatives

A comparative study has been made of the abilities of retinol and its derivatives to induce cell fusion and haemolysis of hen erythrocytes and to cause swelling of rat liver mitochondria. Retinol, retinaldehyde, α-retinoic acid, iso-13-retinol and to a lesser extent retinyl acetate were active in all three systems. Iso-13-retinoic acid was extremely membranolytic but did not produce stable fused cells. By contrast retinoic acid, its cyclopentyl derivative RO8-7699, and the long chain fatty acid esters of retinol, viz. the oleate, linoleate and palmitate esters, were neither fusogenic nor haemolytic, nor did they affect mitochondria.     

CHCHD2 inhibits apoptosis by interacting with Bcl-x L to regulate Bax activation

Mitochondrial outer membrane permeabilization (MOMP) is a critical control point during apoptosis that results in the release of pro-apoptotic mitochondrial contents such as cytochrome c. MOMP is largely controlled by Bcl-2 family proteins such as Bax, which under various apoptotic stresses becomes activated and oligomerizes on the outer mitochondrial membrane. Bax oligomerization helps promote the diffusion of the mitochondrial contents into the cytoplasm activating the caspase cascade. In turn, Bax is regulated primarily by anti-apoptotic Bcl-2 proteins including Bcl-xL, which was recently shown to prevent Bax from accumulating at the mitochondria. However, the exact mechanisms by which Bcl-xL regulates Bax and thereby MOMP remain partially understood. In this study, we show that the small CHCH-domain-containing protein CHCHD2 binds to Bcl-xL and inhibits the mitochondrial accumulation and oligomerization of Bax. Our data show that in response to apoptotic stimuli, mitochondrial CHCHD2 decreases prior to MOMP. Furthermore, when CHCHD2 is absent from the mitochondria, the ability of Bcl-xL to inhibit Bax activation and to prevent apoptosis is attenuated, which results in increases in Bax oligomerization, MOMP and apoptosis. Collectively, our findings establish CHCHD2, a previously uncharacterized small mitochondrial protein with no known homology to the Bcl-2 family, as one of the negative regulators of mitochondria-mediated apoptosis.Apoptosis is a tightly regulated form of programmed cell death that is critical for proper embryonic development, tissue homeostasis and immune response. Aberrant regulation of apoptosis contributes to a wide range of ailments including autoimmune disorders, neurodegenerative diseases and cancer. Unlike necrotic cell death, apoptosis is a genetic program that is characterized by distinct morphological features such as membrane blebbing, chromatin condensation, DNA fragmentation and cell shrinkage.¹ In vertebrates, apoptosis can occur through two pathways: extrinsic, or receptor-mediated apoptosis, and intrinsic, or mitochondria-mediated apoptosis. Intrinsic apoptosis is induced by cellular stressors such as DNA damage, which lead to mitochondrial outer membrane permeabilization (MOMP), cytochrome c release from the mitochondrial intermembrane space, activation of cysteine proteases (caspases) and induction of apoptosis. Once MOMP occurs, cell death is thought to be inevitable. Therefore, much research has been devoted to elucidating the mechanisms and signaling pathways that govern this critical regulatory point in apoptosis.MOMP is controlled largely by the B-cell lymphoma 2 (Bcl-2) family of proteins,² all of which contain at least one of four BH (Bcl-2 homology) domains designated BH1–4. During apoptosis, the pro-apoptotic Bcl-2 proteins Bax and/or Bak become activated and oligomerize on the mitochondrial outer membrane³ increasing mitochondrial membrane permeabilization through a mechanism that is not entirely clear. Bax and Bak are activated by BH3-only Bcl-2 family proteins such as Bim, t-Bid and Puma.^{4, 5, 6, 7, 8, 9, 10, 11, 12, 13} Conversely, Bax and Bak are inhibited by pro-survival Bcl-2 family proteins such as Bcl-2, Mcl-1 and Bcl-xL.^{2, 14, 15, 16} Of the pro-survival Bcl-2 family proteins, Bcl-2 is found at the outer mitochondrial membrane, whereas Bcl-xL and Mcl-1 localize to the outer mitochondrial membrane and the mitochondrial matrix.^{17, 18} Matrix-localized Bcl-xL and Mcl-1 have been shown to promote mitochondrial respiration,¹⁹ suggesting that crosstalk exists between apoptotic pathways and other mitochondria-based biological events. Based on this recent discovery, one might reason that other mitochondrial proteins previously characterized as structural proteins or metabolism-associated enzymes could play an additional intermediate role in the regulation of apoptosis by interacting with Bcl-2 family proteins.We identified CHCHD2 in a mass spectrometry-based screen for binding partners of p32, a mitochondrial protein previously shown by our lab to bind and mediate the apoptotic effects of the tumor suppressor p14ARF.²⁰ CHCHD2 was subsequently detected in independent screens for proteins that regulate cellular metabolism and migration;^{21, 22} however, the functions of CHCHD2 remain unknown. CHCHD2 is encoded by the chchd2 gene (coiled-coil helix coiled-coil helix domain-containing 2), which spans 4921 base pairs, contains 4 exons, and is located on human chromosome 7p11.2, a chromosomal region that is often amplified in glioblastomas.²³ The protein encoded by the chchd2 gene is ubiquitously expressed²⁴ and is relatively small, as it codes for only 151 amino acids. CHCHD2 is well-conserved among different species from humans to yeast, and mouse and human CHCHD2 share 87% amino acid sequence identity (Supplementary Figures S1A and S1B). CHCHD2 contains a C-terminal CHCH (coiled-coil helix coiled-coil helix) domain, which is characterized primarily by four cysteine residues spaced 10 amino acids apart from one another (CX(9)C motif).²⁵ The function of the CHCH domain is not well understood, and the few characterized proteins that harbor this domain have diverse functions. Many CHCH domain-containing proteins localize to the mitochondrial inner membrane or the intermembrane space, including Cox12, Cox17, Cox19, Cox23, Mia40 (yeast homolog of human CHCHD4), CHCHD3 and CHCHD6. Cox17 and Cox19 aid in the assembly of the COX complex,^{26, 27} whereas Mia40/Tim40 has been shown to transport proteins into the mitochondrial intermembrane space.^{28, 29} Furthermore, CHCHD3 and CHCHD6 are essential for maintaining the integrity of mitochondrial cristae and thus mitochondrial function.^{30, 31, 32} Interestingly, a recent report has shown that CHCHD6 is regulated by DNA damage stress, and alterations in CHCHD6 expression affect the viability of breast cancer cells in response to genotoxic anticancer drugs.³²Despite advances in our understanding of how MOMP and apoptosis are regulated by the Bcl-2 family of proteins, much remains unknown with respect to the mechanisms that lead to Bax activation and oligomerization particularly concerning the roles that mitochondria-associated proteins play in the process. In this study, we characterize the small, mitochondria-localized protein CHCHD2 as a novel regulator of Bax oligomerization and apoptosis. Furthermore, we show evidence that CHCHD2 binds to Bcl-xL at the mitochondria under unstressed conditions. In response to apoptotic stimuli, CHCHD2 decreases and loses its mitochondria localization, which is accompanied by decreased Bcl-xL–Bax interaction and increased Bax homo-oligomerization and Bax–Bak hetero-oligomerization. Collectively, our results suggest that CHCHD2 negatively regulates the apoptotic cascade upstream of Bax oligomerization.     

JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation and mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells

Mitochondrial translocation of pro-apoptotic Bax prior to apoptosis is well established after treatment with many cell death stimulants or under apoptosis-inducing conditions. The mechanism of mitochondrial translocation of Bax is, however, still unknown. The aim of this work was to investigate the mechanism of Bax activation and mitochondrial translocation to initiate apoptosis of human hepatoma HepG2 and porcine kidney LLC-PK1 cells exposed to various cell death agonists. Phosphorylation of Bax by JNK and p38 kinase activated after treatment with staurosporine, H(2)O(2), etoposide, and UV light was demonstrated by the shift in the pI value of Bax on two-dimensional gels and confirmed by metabolic labeling with inorganic [(32)P]phosphate in HepG2 cells. Specific inhibitors of JNK and p38 kinase significantly inhibited Bax phosphorylation and mitochondrial translocation and apoptosis of HepG2 cells. A specific small interfering RNA to MAPKK4 (the upstream protein kinase of JNK and p38 kinase) markedly decreased the levels of MAPKK4 and MAPKK3/6, blocked the activation of JNK or p38 kinase, and inhibited Bax phosphorylation. However, the negative control small interfering RNA did not cause these changes. Confocal microscopy of various Bax mutants showed differential rates of mitochondrial translocation of Bax before and after staurosporine treatment. Among the Bax mutants, T167D did not translocate to mitochondria after staurosporine exposure, suggesting that Thr(167) is a potential phosphorylation site. In conclusion, our results demonstrate, for the first time, that Bax is phosphorylated by stress-activated JNK and/or p38 kinase and that phosphorylation of Bax leads to mitochondrial translocation prior to apoptosis.     

Mitochondrial clustering induced by overexpression of the mitochondrial fusion protein Mfn2 causes mitochondrial dysfunction and cell death

Mitochondria change their shapes dynamically mainly through fission and fusion. Dynamin-related GTPases have been shown to mediate remodeling of mitochondrial membranes during these processes. One of these GTPases, mitofusin, is anchored at the outer mitochondrial membrane and mediates fusion of the outer membrane. We found that overexpression of a mitofusin isoform, Mfn2, drastically changes mitochondrial morphology, forming mitochondrial clusters. High-resolution microscopic examination indicated that the mitochondrial clusters consisted of small fragmented mitochondria. Inhibiting mitochondrial fission prevented the cluster formation, supporting the notion that mitochondrial clusters are formed by fission-mediated mitochondrial fragmentation and aggregation. Mitochondrial clusters displayed a decreased inner membrane potential and mitochondrial function, suggesting a functional compromise of small fragmented mitochondria produced by Mfn2 overexpression; however, mitochondrial clusters still retained mitochondrial DNA. We found that cells containing clustered mitochondria lost cytochrome c from mitochondria and underwent caspase-mediated apoptosis. These results demonstrate that mitochondrial deformation impairs mitochondrial function, leading to apoptotic cell death and suggest the presence of an intricate form-function relationship in mitochondria.     

PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation

A change in the metabolic flux of glucose from mitochondrial oxidative phosphorylation (OXPHOS) to aerobic glycolysis is regarded as one hallmark of cancer. However, the mechanisms underlying the metabolic switch between aerobic glycolysis and OXPHOS are unclear. Here we show that the M2 isoform of pyruvate kinase (PKM2), one of the rate-limiting enzymes in glycolysis, interacts with mitofusin 2 (MFN2), a key regulator of mitochondrial fusion, to promote mitochondrial fusion and OXPHOS, and attenuate glycolysis. mTOR increases the PKM2:MFN2 interaction by phosphorylating MFN2 and thereby modulates the effect of PKM2: MFN2 on glycolysis, mitochondrial fusion and OXPHOS. Thus, an mTOR-MFN2-PKM2 signaling axis couples glycolysis and OXPHOS to modulate cancer cell growth.     

Spatial and temporal association of Bax with mitochondrial fission sites,Drp1, and Mfn2 during apoptosis

We find that Bax, a proapoptotic member of the Bcl-2 family, translocates to discrete foci on mitochondria during the initial stages of apoptosis, which subsequently become mitochondrial scission sites. A dominant negative mutant of Drp1, Drp1K38A, inhibits apoptotic scission of mitochondria, but does not inhibit Bax translocation or coalescence into foci. However, Drp1K38A causes the accumulation of mitochondrial fission intermediates that are associated with clusters of Bax. Surprisingly, Drp1 and Mfn2, but not other proteins implicated in the regulation of mitochondrial morphology, colocalize with Bax in these foci. We suggest that Bax participates in apoptotic fragmentation of mitochondria.     

Increase in cytosolic Ca2+ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes

Reactive oxygen species are important regulators of protozoal infection. Promastigotes of Leishmania donovani, the causative agent of Kala-azar, undergo an apoptosis-like death upon exposure to H2O2. The present study shows that upon activation of death response by H2O2, a dose- and time-dependent loss of mitochondrial membrane potential occurs. This loss is accompanied by a depletion of cellular glutathione, but cardiolipin content or thiol oxidation status remains unchanged. ATP levels are reduced within the first 60 min of exposure as a result of mitochondrial membrane potential loss. A tight link exists between changes in cytosolic Ca2+ homeostasis and collapse of the mitochondrial membrane potential, but the dissipation of the potential is independent of elevation of cytosolic Na+ and mitochondrial Ca2+. Partial inhibition of cytosolic Ca2+ increase achieved by chelating extracellular or intracellular Ca2+ by the use of appropriate agents resulted in significant rescue of the fall of the mitochondrial membrane potential and apoptosis-like death. It is further demonstrated that the increase in cytosolic Ca2+ is an additive result of release of Ca2+ from intracellular stores as well as by influx of extracellular Ca2+ through flufenamic acid-sensitive non-selective cation channels; contribution of the latter was larger. Mitochondrial changes do not involve opening of the mitochondrial transition pore as cyclosporin A is unable to prevent mitochondrial membrane potential loss. An antioxidant like N-acetylcysteine is able to inhibit the fall of the mitochondrial membrane potential and prevent apoptosis-like death. Together, these findings show the importance of non-selective cation channels in regulating the response of L. donovani promastigotes to oxidative stress that triggers downstream signaling cascades leading to apoptosis-like death.     

BARD1 translocation to mitochondria correlates with Bax oligomerization, loss of mitochondrial membrane potential, and apoptosis

The breast cancer regulatory protein-1 (BRCA1)-associated RING domain 1 (BARD1) gene is mutated in a subset of breast/ovarian cancers. BARD1 functions as a heterodimer with BRCA1 in nuclear DNA repair. BARD1 also has a BRCA1-independent apoptotic activity. Here we investigated the link between cytoplasmic localization and apoptotic function of BARD1. We used immunofluorescence microscopy and deconvolution analysis to resolve BARD1 cytoplasmic staining patterns and detected endogenous BARD1 at mitochondria. BARD1 was also detected in mitochondrial cell fractions by immunoblotting. The targeting of BARD1 to mitochondria was modestly stimulated by DNA damage and did not require BRCA1 as indicated by RNA interference and peptide-competition experiments. Transiently expressed yellow fluorescence protein-BARD1 localized to mitochondria, and the targeting sequences were mapped to both the N and C terminus of BARD1. Ectopic yellow fluorescence protein-BARD1 induced apoptosis and loss of mitochondrial membrane potential in MCF-7 breast tumor cells. BARD1 apoptotic function was associated with stimulation of Bax oligomerization at mitochondria. This distinguishes it from BRCA1, which is pro-apoptotic but did not induce Bax oligomerization. The cancer-associated BARD1 splice-variant DeltaRIN (lacks the BRCA1 binding domain and ankyrin repeats) was recruited to mitochondria but did not stimulate apoptosis or alter membrane permeability. We propose that BARD1 has two main sites of action in its cellular response to DNA damage, the nucleus, where it promotes cell survival through DNA repair, and the mitochondria, where BARD1 regulates apoptosis.     

LTB4 induced activation signals and responses in neutrophils are short-lived compared to formylpeptide

Leukotriene B4 (LTB4) was shown to be a potent stimulator of neutrophil actin polymerization as detected by right-angle light scatter and rhodamine-phalloidin staining of F-actin. When we compared the kinetics of this neutrophil cytoskeletal response to the chemoattractants formylpeptide and LTB4, we observed that the response to LTB4 was markedly shorter-lived. To understand the basis of this result, we re-examined the kinetics of superoxide generation, elastase release, intracellular calcium elevation, and phosphoinositide metabolism in neutrophils stimulated with LTB4 and N-formylhexapeptide. LTB4 was relatively inefficient in producing superoxide and in releasing elastase. Although both responses were initiated with similar rapidity, they turned off sooner with LTB4 as compared with N-formylhexapeptide stimulation. Intracellular calcium elevation, a signal that is necessary for superoxide generation and degranulation, was of similar magnitude but of shorter duration in response to LTB4 as compared with the N-formylhexapeptide. The LTB4-induced rise of phosphatidic acid also was not sustained as long as the N-formylhexapeptide-induced increase. Prior exposure of neutrophils to LTB4 did not inhibit subsequent stimulation of superoxide generation by N-formylhexapeptide. Thus, the inability of LTB4 to stimulate superoxide generation was not due to LTB4-induced global inhibitory signals. The deficiency in LTB4-induced superoxide and elastase responses may be related to short-lived LTB4-induced activation signals and/or the number of receptors contributing to these responses.     

Arachidonic acid enhances intracellular [Ca2+]i increase and mitochondrial depolarization induced by glutamate in cerebellar granule cells

Maturation of primary neuronal cultures is accompanied by an increase in the proportion of cells that exhibit biphasic increase in free cytoplasmic Ca2+ ([Ca2+]i) followed by synchronic decrease in electrical potential difference across the inner mitochondrial membrane (DeltaPsim) in response to stimulation of glutamate receptors. In the present study we have examined whether the appearance of the second phase of [Ca2+]i change can be attributed to arachidonic acid (AA) release in response to the effect of glutamate (Glu) on neurons. Using primary culture of rat cerebellar granule cells we have investigated the effect of AA (1-20 microM) on [Ca2+]i, DeltaPsim, and [ATP] and changes in these parameters induced by neurotoxic concentrations of Glu (100 microM, 10-40 min). At =10 microM, AA caused insignificant decrease in DeltaPsim without any influence on [Ca2+]i. The mitochondrial ATPase inhibitor oligomycin enhanced AA-induced decrease in DeltaPsim; this suggests that AA may inhibit mitochondrial respiration. Addition of AA during the treatment with Glu resulted in more pronounced augmentation of [Ca2+]i and the decrease in DeltaPsim than the changes in these parameters observed during independent action of AA; removal of Glu did not abolish these changes. An inhibitor of the cyclooxygenase and lipoxygenase pathways of AA metabolism, 5,8,11,14-eicosatetraynoic acid, increased the proportion of neurons characterized by Glu-induced biphasic increase in [Ca2+]i and the decrease in DeltaPsim. Palmitic acid (30 microM) did not increase the percentage of neurons exhibiting biphasic response to Glu. Co-administration of AA and Glu caused 2-3 times more pronounced decrease in ATP concentrations than that observed during the independent effect of AA and Glu. The data suggest that AA may influence the functional state of mitochondria, and these changes may promote biphasic [Ca2+]i and DeltaPsim responses of neurons to the neurotoxic effect of Glu.     

Kaempferol induces apoptosis in two different cell lines via Akt inactivation,Bax and SIRT3 activation,and mitochondrial dysfunction

Kaempferol (3,4′,5,7‐tetrahydroxyflavone) is a flavonoid with anti‐ and pro‐oxidant activity present in various natural sources. Kaempferol has been shown to posses anticancer properties through the induction of the apoptotic program. Here we report that treatment of the chronic myelogenous leukemia cell line K562 and promyelocitic human leukemia U937 with 50 µM kaempferol resulted in an increase of the antioxidant enzymes Mn and Cu/Zn superoxide dismutase (SOD). Kaempferol treatment induced apoptosis by decreasing the expression of Bcl‐2 and increasing the expressions of Bax. There were also induction of mitochondrial release of cytochrome c into cytosol and significant activation of caspase‐3, and ‐9 with PARP cleavage. Kaempferol treatment increased the expression and the mitochondria localization of the NAD‐dependent deacetylase SIRT3. K562 cells stably overexpressing SIRT3 were more sensitive to kaempferol, whereas SIRT3 silencing did not increase the resistance of K562 cells to kaempferol. Inhibition of PI3K and de‐phosphorylation of Akt at Ser473 and Thr308 was also observed after treating both K562 and U937 cells with kaempferol. In conclusion our study shows that the oxidative stress induced by kaempferol in K562 and U937 cell lines causes the inactivation of Akt and the activation of the mitochondrial phase of the apoptotic program with an increase of Bax and SIRT3, decrease of Bcl‐2, release of cytochrome c, caspase‐3 activation, and cell death. J. Cell. Biochem. 106: 643–650, 2009. © 2009 Wiley‐Liss, Inc.