Mitochondrial modulation-induced activation of vagal sensory neuronal subsets by antimycin A, but not CCCP or rotenone, correlates with mitochondrial superoxide production.

Inflammation causes nociceptive sensory neuron activation, evoking debilitating symptoms and reflexes. Inflammatory signaling pathways are capable of modulating mitochondrial function, resulting in reactive oxygen species (ROS) production, mitochondrial depolarization and calcium release. Previously we showed that mitochondrial modulation with antimycin A, a complex III inhibitor, selectively stimulated nociceptive bronchopulmonary C-fibers via the activation of transient receptor potential (TRP) ankyrin 1 (A1) and vanilloid 1 (V1) cation channels. TRPA1 is ROS-sensitive, but there is little evidence that TRPV1 is activated by ROS. Here, we used dual imaging of dissociated vagal neurons to investigate the correlation of mitochondrial superoxide production (mitoSOX) or mitochondrial depolarization (JC-1) with cytosolic calcium (Fura-2AM), following mitochondrial modulation by antimycin A, rotenone (complex I inhibitor) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP, mitochondrial uncoupling agent). Mitochondrial modulation by all agents selectively increased cytosolic calcium in a subset of TRPA1/TRPV1-expressing (A1/V1+) neurons. There was a significant correlation between antimycin A-induced calcium responses and mitochondrial superoxide in wild-type 'responding' A1/V1+ neurons, which was eliminated in TRPA1-/- neurons, but not TRPV1-/- neurons. Nevertheless, antimycin A-induced superoxide production did not always increase calcium in A1/V1+ neurons, suggesting a critical role of an unknown factor. CCCP caused both superoxide production and mitochondrial depolarization but neither correlated with calcium fluxes in A1/V1+ neurons. Rotenone-induced calcium responses in 'responding' A1/V1+ neurons correlated with mitochondrial depolarization but not superoxide production. Our data are consistent with the hypothesis that mitochondrial dysfunction causes calcium fluxes in a subset of A1/V1+ neurons via ROS-dependent and ROS-independent mechanisms.

Differential effects of mitochondrial inhibitors on porcine granulosa cells and oocytes.

Oocytes and granulosa cells rely primarily on mitochondrial respiration and glycolysis for energy production, respectively. The present study examined the effect of mitochondrial inhibitors on the ATP contents of oocytes and granulosa cells. Cumulus cell-oocyte complexes (COCs) and granulosa cells (GCs) were collected from the antral follicles of porcine ovaries. Treatment of denuded oocytes with either carbonyl cyanide m-chlorophenyl hydrazine (CCCP), antimycin, or oligomycin significantly reduced ATP content to very low levels (CCCP, 0.12 pM; antimycin, 0.07 pM; and oligomycin, 0.25 pM; P < 0.05), whereas treatment with a glycolysis inhibitor (bromopyruvic acid, BA) had no effect. Conversely, the ATP content of granulosa cells was significantly reduced by treatment with the glycolysis inhibitor but was not affected by the mitochondrial inhibitors (ATP/10,000 cells; control, 1.78 pM and BA, 0.32 pM; P < 0.05). Reactive oxygen species (ROS) generation after CCCP treatment was greater in oocytes (1.6-fold) than that seen in granulosa cells (1.08-fold). Oocytes surrounded by granulosa cells had higher ATP levels than denuded oocytes. Treatment of COCs with CCCP reduced, but did not completely abolish, ATP content in oocytes (control, 3.15 pM and CCCP, 0.52 pM; P < 0.05), whereas treatment with CCCP plus a gap junction inhibitor, 18α-glycyrrhetinic acid, and CCCP decreased the ATP content to even lower levels (0.29 pM; P < 0.05). These results suggest that granulosa cells are dependent on glycolysis and provide energy to oocytes through gap junctions, even after treatment with CCCP.

Expression of the alternative oxidase mitigates beta-amyloid production and toxicity in model systems.

Mitochondrial dysfunction has been widely associated with the pathology of Alzheimer's disease, but there is no consensus on whether it is a cause or consequence of disease, nor on the precise mechanism(s). We addressed these issues by testing the effects of expressing the alternative oxidase AOX from Ciona intestinalis, in different models of AD pathology. AOX can restore respiratory electron flow when the cytochrome segment of the mitochondrial respiratory chain is inhibited, supporting ATP synthesis, maintaining cellular redox homeostasis and mitigating excess superoxide production at respiratory complexes I and III. In human HEK293-derived cells, AOX expression decreased the production of beta-amyloid peptide resulting from antimycin inhibition of respiratory complex III. Because hydrogen peroxide was neither a direct product nor substrate of AOX, the ability of AOX to mimic antioxidants in this assay must be indirect. In addition, AOX expression was able to partially alleviate the short lifespan of Drosophila models neuronally expressing human beta-amyloid peptides, whilst abrogating the induction of markers of oxidative stress. Our findings support the idea of respiratory chain dysfunction and excess ROS production as both an early step and as a pathologically meaningful target in Alzheimer's disease pathogenesis, supporting the concept of a mitochondrial vicious cycle underlying the disease.

Identification of interacting partners of Human Mpv17-like protein with a mitigating effect of mitochondrial dysfunction through mtDNA damage.

Human Mpv17-like protein (M-LPH) has been suggested to participate in mitochondrial function. In this study, we investigated the proteins that interact with M-LPH, and identified four: H2A histone family, member X (H2AX), ribosomal protein S14 (RPS14), ribosomal protein S3 (RPS3) and B-cell receptor-associated protein 31 (Bap31). Immunofluorescence and subcellular fractionation studies revealed that M-LPH is localized predominantly in the nucleus, to some extent in a subset of mitochondria, and marginally in the cytosol. Mitochondrial M-LPH appeared as punctate foci, and these were co-localized with a subset of mitochondrial transcription factor A (TFAM) and mtDNA, indicating that M-LPH is localized in or in close proximity to mitochondrial nucleoids. RNAi-mediated knockdown of M-LPH resulted in an increase of mtDNA damage and reduced the expression of mtDNA-encoded genes. A ROS inducer, antimycin A, caused an increase in both the number and size of the mitochondrial M-LPH foci, and these foci were co-localized with two enzymes, DNA polymerase γ (POLG) and DNA ligase III (LIG3), both involved in mtDNA repair. Furthermore, knockdown of M-LPH hampered mitochondrial localization of these enzymes. Taken together, these observations suggest that M-LPH is involved in the maintenance of mtDNA and protects cells from mitochondrial dysfunction.

Alpha7 nicotinic receptor activation protects against oxidative stress via heme-oxygenase I induction.

Subchronic oxidative stress and inflammation are being increasingly implicated in the pathogenesis of numerous diseases, such as Alzheimer's or Parkinson's disease. This study was designed to evaluate the potential protective role of α7 nicotinic receptor activation in an in vitro model of neurodegeneration based on subchronic oxidative stress. Rat organotypic hippocampal cultures (OHCs) were exposed for 4 days to low concentration of lipopolysaccharide (LPS) and the complex III mitochondrial blocker, antimycin-A. Antimycin-A (0.1µM) and lipopolysaccharide (1ng/ml) caused low neurotoxicity on their own, measured as propidium iodide fluorescence in CA1 and CA3 regions. However, their combination (LPS/AA) caused a greater detrimental effect, in addition to mitochondrial depolarization, overproduction of reactive oxygen species (ROS) and Nox4 overexpression. Antimycin-A per se increased ROS and mitochondrial depolarization, although these effects were significantly higher when combined with LPS. More interesting was the finding that exposure of OHCs to the combination of LPS/AA triggered aberrant protein aggregation, measured as thioflavin S immunofluorescence. The α7 nicotinic receptor agonist, PNU282987, prevented the neurotoxicity and the pathological hallmarks observed in the LPS/AA subchronic toxicity model (oxidative stress and protein aggregates); these effects were blocked by α-bungarotoxin and tin protoporphyrin, indicating the participation of α7 nAChRs and heme-oxygenase I induction. In conclusion, subchronic exposure of OHCs to low concentration of antimycin-A plus LPS reproduced pathological features of neurodegenerative disorders. α7 nAChR activation ameliorated these alterations by a mechanism involving heme-oxygenase I induction.

Uncoupling protein-2 attenuates palmitoleate protection against the cytotoxic production of mitochondrial reactive oxygen species in INS-1E insulinoma cells.

High glucose and fatty acid levels impair pancreatic beta cell function. We have recently shown that palmitate-induced loss of INS-1E insulinoma cells is related to increased reactive oxygen species (ROS) production as both toxic effects are prevented by palmitoleate. Here we show that palmitate-induced ROS are mostly mitochondrial: oxidation of MitoSOX, a mitochondria-targeted superoxide probe, is increased by palmitate, whilst oxidation of the equivalent non-targeted probe is unaffected. Moreover, mitochondrial respiratory inhibition with antimycin A stimulates palmitate-induced MitoSOX oxidation. We also show that palmitate does not change the level of mitochondrial uncoupling protein-2 (UCP2) and that UCP2 knockdown does not affect palmitate-induced MitoSOX oxidation. Palmitoleate does not influence MitoSOX oxidation in INS-1E cells ±UCP2 and largely prevents the palmitate-induced effects. Importantly, UCP2 knockdown amplifies the preventive effect of palmitoleate on palmitate-induced ROS. Consistently, viability effects of palmitate and palmitoleate are similar between cells ±UCP2, but UCP2 knockdown significantly augments the palmitoleate protection against palmitate-induced cell loss at high glucose. We conclude that UCP2 neither mediates palmitate-induced mitochondrial ROS generation and the associated cell loss, nor protects against these deleterious effects. Instead, UCP2 dampens palmitoleate protection against palmitate toxicity.

P-glycoprotein mediates the collateral sensitivity of multidrug resistant cells to steroid hormones.

The overexpression of P-glycoprotein (P-gp, ABCB1) in cancer cells often leads to multidrug resistance (MDR) through reduced drug accumulation. However, certain P-gp-positive cells display hypersensitivity, or collateral sensitivity, to certain compounds that are believed to induce Pgp-dependent oxidative stress. We have previously reported that MDR P-gp-positive CHO cells are collaterally sensitive to verapamil (VRP; Laberge et al. (2009) [1]). In this report we extend our previous findings and show that drug resistant CHO cells are also collaterally sensitive to physiologic levels of progesterone (PRO) and deoxycorticosterone (DOC). Both PRO and DOC collateral sensitivities in CH(R)C5 cells are dependent on P-gp-expression and ATPase, as knockdown of P-gp expression with siRNA or inhibition of P-gp-ATPase with PSC833 reverses PRO- and DOC-induced collateral sensitivity. Moreover, the mitochondrial complexes I and III inhibitors (antimycin-A and rotenone, respectively) synergize with PRO and DOC-induced collateral sensitivity. We also show that VRP inhibits PRO and DOC collateral sensitivity, consistent with earlier findings relating to the VRP's modulation of PRO and DOC-stimulation of P-gp ATPase. The findings of this study demonstrate a P-gp-dependent collateral sensitivity of MDR cells in the presence of physiologically achievable concentrations of progesterone and deoxycorticosterone.

Antimycin A sensitizes cells to TRAIL-induced apoptosis through upregulation of DR5 and downregulation of c-FLIP and Bcl-2.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been the focus as a potential anticancer drug, because it induces apoptosis in a wide variety of cancer cells but not in most normal human cell types. In this study, we showed that combination treatment with sub-toxic doses of antimycin A (AMA), an inhibitor of electron transport, plus TRAIL induced apoptosis in human renal cancer cells, but not in normal tubular kidney cells. Treatment of Caki cells with AMA upregulated the death receptor 5 (DR5) protein and downregulated c-FLIP and Bcl-2 proteins in a dose-dependent manner. AMA-induced decrease of c-FLIPL and c-FLIPs protein levels which were caused by increased protein instability, which was confirmed by the result showing that treatment with a protein biosynthesis inhibitor, CHX, accelerated degradation of c-FLIPL and c-FLIPs proteins caused by AMA treatment. We also found that AMA induced upregulation of DR5 and downregulation of Bcl-2 at the transcriptional level. Pretreatment with N-acetyl-l-cysteine (NAC) partly recovered the expression levels of c-FLIPL and c-FLIPs proteins were downregulated by the AMA treatment, suggesting that AMA appears to be partially dependent on the generation of ROS for downregulation of c-FLIPL and c-FLIPs. Collectively, this study demonstrates that AMA enhances TRAIL-induced apoptosis in human renal cancer cells by upregulation of DR5 as well as downregulation of c-FLIP and Bcl-2. Furthermore, this study shows that AMA markedly increases sensitivity to cisplatin in Caki human renal cancer cells.

Ginkgo extract EGb 761(®) shields from slowly accumulating neurodegenerative-like changes in a newly developed cell culture model induced by the combined action of low doses of antimycin A1 and 2-deoxy-D-glucose.

Different cell culture models were already used to analyze the molecular base of the neuroprotective activities of the Ginkgo biloba extract EGb 761(®) after a single or short-term application. In these previous studies cells were severely injured with agents that promptly induce fatal cellular damage, like vast oxidative stress or mitochondrial dysfunction, and the protective effects of EGb 761(®) on such acute damage were evaluated. Our present study aimed to test EGb 761(®) action in cell cultures, where cellular functions are only moderately impaired by a longer lasting, but relatively modest oxidative stress, reduction of mitochondrial function and reduced intracellular energy levels, thereby causing only slow occurence of cellular damage over a time period of 2 weeks. To this end we used neuroblastoma cells (SK-N-MC) that were treated with low doses of a combination of antimycin A1 and 2-deoxy-D: -glucose. Addition of EGb 761(®) to the culture medium efficiently shielded the cells from progressing injury by reduced ATP-levels, oxidized redox state, lipid peroxidation damage and oxidative damage of mitochondrial DNA. As a result the cells were protected from apoptotic death that was observed in cultures without EGb 761(®) after 2 weeks of damage occurence. This cell culture system characterizing moderate cellular stress will be applied in future studies to further investigate the mode of action of single EGb 761(®) compounds.

Simple isolation of antimycin A1 and some of its toxicological properties.

An unidentified actinomycete, RTI 246, was found to produce antimycin A(1) in high yield on a high protein cereal medium. The antibiotic compound was extracted from the cells and isolated in pure form by crystallization. It was identified by ultraviolet, infrared, nuclear magnetic resonance, and mass spectroscopy and by alkaline hydrolysis to antimycic acid and a neutral lactone. The intravenous LD(50) was 1.0 mg/kg in white mice, whereas the intraperitoneal LD(50) was 1.50 +/- 0.19 mg/kg. Animals receiving an intraperitoneal injection displayed an incoordination of the hind limbs and impaired reflexes before showing signs of respiratory distress. These findings indicated that antimycin A(1) possesses a neurotoxic property separate from its well-documented property as a respiratory poison.