Voltage-dependent anion channel (VDAC) as mitochondrial governator—Thinking outside the box

Despite a detailed understanding of their metabolism, mitochondria often behave anomalously. In particular, global suppression of mitochondrial metabolism and metabolite exchange occurs in apoptosis, ischemia and anoxia, cytopathic hypoxia of sepsis and multiple organ failure, alcoholic liver disease, aerobic glycolysis in cancer cells (Warburg effect) and unstimulated pancreatic beta cells. Here, we propose that closure of voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane accounts for global mitochondrial suppression. In anoxia, cytopathic hypoxia and ethanol treatment, reactive oxygen and nitrogen species, cytokines, kinase cascades and increased NADH act to inhibit VDAC conductance and promote selective oxidation of membrane-permeable respiratory substrates like short chain fatty acids and acetaldehyde. In cancer cells, highly expressed hexokinase binds to and inhibits VDAC to suppress mitochondrial function while stimulating glycolysis, but an escape mechanism intervenes when glucose-6-phosphate accumulates and dissociates hexokinase from VDAC. Similarly, glucokinase binds mitochondria of insulin-secreting beta cells, possibly blocking VDAC and suppressing mitochondrial function. We propose that glucose metabolism leads to glucose-6-phosphate-dependent unbinding of glucokinase, relief of VDAC inhibition, release of ATP from mitochondria and ATP-dependent insulin release. In support of the overall proposal, ethanol treatment of isolated rat hepatocytes inhibited mitochondrial respiration and accessibility to adenylate kinase in the intermembrane space, effects that were overcome by digitonin permeabilization of the outer membrane. Overall, these considerations suggest that VDAC is a dynamic regulator, or governator, of global mitochondrial function both in health and disease

Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 μM digitonin. Blockade of VDAC with Koenig's polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 μM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC

Closure of VDAC causes oxidative stress and accelerates the Ca2+-induced mitochondrial permeability transition in rat liver mitochondria

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca2+-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O2•-) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O2•- from the IMS and sensitizing to the Ca2+-induced MPT. Treatment of isolated rat liver mitochondria with 5 µM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8 ± 1.4 min within a range of 100–250 µM Ca2+. G3139-mediated acceleration of the MPT was reversed by 20 µM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O2•- in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O2•- accumulation. Mathematical modeling of generation and turnover of O2•- within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O2•-. In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O2•-˜, which causes an internal oxidative stress and sensitizes mitochondria toward the Ca2+-induced MPT

Bactericidal Efficacy of Nitric Oxide-Releasing Silica Nanoparticles

The utility of nitric oxide (NO)-releasing silica nanoparticles as a novel antibacterial is demonstrated against Pseudomonas aeruginosa. Nitric oxide-releasing nanoparticles were prepared via co-condensation of tetraalkoxysilane with aminoalkoxysilane modified with diazeniumdiolate NO donors, allowing for the storage of large NO payloads. Comparison of the bactericidal efficacy of the NO-releasing nanoparticles to 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO), a small molecule NO donor, demonstrated enhanced bactericidal efficacy of nanoparticle-derived NO and reduced cytotoxicity to healthy cells (mammalian fibroblasts). Confocal microscopy revealed that fluorescently-labeled NO-releasing nanoparticles associated with the bacteria, providing rationale for the enhanced bactericidal efficacy of the nanoparticles. Intracellular NO concentrations were measurable when the NO was delivered from nanoparticles as opposed to PROLI/NO. Collectively, these results demonstrate the advantage of delivering NO via nanoparticles for antimicrobial applications

Minocycline and N-methyl-4-isoleucine cyclosporin (NIM811) mitigate storage/reperfusion injury after rat liver transplantation through suppression of the mitochondrial permeability transition

Graft failure after liver transplantation may involve mitochondrial dysfunction. We examined whether prevention of mitochondrial injury would improve graft function. Orthotopic rat liver transplantation was performed after 18 hours' cold storage in University of Wisconsin solution and treatment with vehicle, minocycline, tetracycline, or N-methyl-4-isoleucine cyclosporin (NIM811) of explants and recipients. Serum alanine aminotransferase (ALT), necrosis, and apoptosis were assessed 6 hours after implantation. Mitochondrial polarization and cell viability were assessed by intravital microscopy. Respiration and the mitochondrial permeability transition (MPT) were assessed in isolated rat liver mitochondria. After transplantation with vehicle or tetracycline, ALT increased to 5242 U/L and 4373 U/L, respectively. Minocycline and NIM811 treatment decreased ALT to 2374 U/L and 2159 U/L, respectively (P < 0.01). Necrosis and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) also decreased from 21.4% and 21 cells/field, respectively, after vehicle to 10.1% and 6 cells/field after minocycline and to 8.7% and 5.2 cells/field after NIM811 (P < 0.05). Additionally, minocycline decreased caspase-3 activity in graft homogenates (P < 0.05). Long-term graft survival was 27% and 33%, respectively, after vehicle and tetracycline treatment, which increased to 60% and 70% after minocycline and NIM811 (P < 0.05). In isolated mitochondria, minocycline and NIM811 but not tetracycline blocked the MPT. Minocycline blocked the MPT by decreasing mitochondrial Ca2+ uptake, whereas NIM811 blocks by interaction with cyclophilin D. Intravital microscopy showed that minocycline and NIM811 preserved mitochondrial polarization and cell viability after transplantation (P < 0.05)

13C magnetic resonance spectroscopy detection of changes in serine isotopomers reflects changes in mitochondrial redox status

The glycine cleavage system (GCS), the major pathway of glycine catabolism in liver, is found only in the mitochondria matrix and is regulated by the NAD+/NADH ratio. In conjunction with serine hydroxymethyltransferase, glycine forms the 1 and 2 positions of serine, while the 3 position is formed exclusively by GCS. Therefore, we sought to exploit this pathway to show that quantitative measurements of serine isotopomers in liver can be used to monitor the NAD+/NADH ratio using 13C NMR spectroscopy. Rat hepatocytes were treated with modulators of GCS activity followed by addition of 2-13C-glycine, and the changes in the proportions of newly synthesized serine isotopomers were compared to controls. Cysteamine, a competitive inhibitor of GCS, prevented formation of mitochondrial 3-13C-serine and 2,3-13C-serine isotopomers while reducing 2-13C-serine by 55%, demonstrating that ca 20% of glycine-derived serine is produced in the cytosol. Glucagon, which activates GCS activity, and the mitochondrial uncoupler carbonyl cyanide-3-chlorophenylhydrazone (CCCP) both increased serine isotopomers, while rotenone, an inhibitor of complex I, had the opposite effect. These results demonstrate that 13C MRS monitoring of the formation of serine isotopomers in isolated rat hepatocytes given 2-13C-glycine reflects the changes of mitochondrial redox status

Inhibition of Mitochondrial Respiration as a Source of Adaphostin-induced Reactive Oxygen Species and Cytotoxicity

Adaphostin is a dihydroquinone derivative that is undergoing extensive preclinical testing as a potential anticancer drug. Previous studies have suggested that the generation of reactive oxygen species (ROS) plays a critical role in the cytotoxicity of this agent. In this study, we investigated the source of these ROS. Consistent with the known chemical properties of dihydroquinones, adaphostin simultaneously underwent oxidation to the corresponding quinone and generated ROS under aqueous conditions. Interestingly, however, this quinone was not detected in intact cells. Instead, high performance liquid chromatography demonstrated that adaphostin was concentrated by up to 300-fold in cells relative to the extracellular medium and that the highest concentration of adaphostin (3000-fold over extracellular concentrations) was detected in mitochondria. Consistent with a mitochondrial site for adaphostin action, adaphostin-induced ROS production was diminished by >75% in MOLT-4 rho(0) cells, which lack mitochondrial electron transport, relative to parental MOLT-4 cells. In addition, inhibition of oxygen consumption was observed when intact cells were treated with adaphostin. Loading of isolated mitochondria to equivalent adaphostin concentrations caused inhibition of uncoupled oxygen consumption in mitochondria incubated with the complex I substrates pyruvate and malate or the complex II substrate succinate. Further analysis demonstrated that adaphostin had no effect on pyruvate or succinate dehydrogenase activity. Instead, adaphostin inhibited reduced decylubiquinone-induced cytochrome c reduction, identifying complex III as the site of inhibition by this agent. Moreover, adaphostin enhanced the production of ROS by succinate-charged mitochondria. Collectively, these observations demonstrate that mitochondrial respiration rather than direct redox cycling of the hydroquinone moiety is a source of adaphostin-induced ROS and identify complex III as a potential target for antineoplastic agents

Mitochondrial Protease ClpP is a Target for the Anticancer Compounds ONC201 and Related Analogues

ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be ∼50-100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with ∼10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a ∼10-100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds

A novel high throughput approach for quantification of cell density

Background: Current approach to cell counting using hemacytometer is limited by requirement for high cell concentration and is prone to error. In biological experiments using cells from human cardiac tissues with limited number of cells, this approach results in large variation in cell counts. Here, we demonstrate the utility of a novel approach using a 96-well microplate that accurately provides the density of cells as low as 15,000 cells/cm2, which fulfills an unmet need in experiments with limited cell availability. Purpose: To develop and test the accuracy of a high-throughput 96-well microplate assay in assessing the cell density in comparison to existing methods. Methods: NIH/3T3 fibroblasts were cultured and differentiated and grown to different cell density. Cell number obtained using hemacytometer was compared to the total fluorescence of propidium iodide, binding to the nuclei of cells permeabilized with Triton X-100 (0.25%), and assessed using multiplate reader. In addition, the total activity of lactate dehydrogenase, an intracellular enzyme, was used to assess the total volume of cytoplasm released from permeabilized cells. Furthermore, the ratio of live/ dead cells was determined by propidium iodide-positive cells and lactate dehydrogenase activity before and after permeabilization in each well of the 96-well plate. Results:There was a linear relationship between increasing intensity of propidium iodide fluorescence with the density of the cells in the 96-well microplate (ranging from 5,000 to 100,000 cells/cm2). Similarly, linear relationship was observed between the intensity of propidium iodide fluorescence and cellular lactate dehydrogenase activity in corresponding wells. At low cell density ( Conclusion: Proposed propidium iodide and lactate dehydrogenase assays are useful tools for quantification of cell number in high-throughput manner with greater accuracy at low cell density, higher reproducibility and overall time saving. This assay is especially useful in experiments using limited cell number such as cells isolated from the human heart

A novel sulfhydryl-sensing fluorescent probe to monitor the redox status of intracellular compartments

Background: Sensing of intracellular redox state is important for detecting the effect of disease and therapeutics agents. However, a reliable assay that can simultaneously provide information about the redox state of intracellular compartments is currently not available. Such an assay will improve the detection of abnormal metabolic state and evaluate the impact of therapeutics. Purpose: To test the responsiveness of DSSQ1 (fluorescein- Donor tethered via a disulfide [S-S] to a para-methyl red Quencher), a novel sulfhydryl-sensing fluorescence probe, and monitor its intracellular distribution under oxidative and reduced conditions. Methods: Fibroblasts grown in culture were treated with redox sensor DSSQ1, and its intracellular distribution and localization was assessed using confocal fluorescent microscopy. Localization of DSSQ1 within mitochondria, lysosomes and nuclei was confirmed using specific fluorescent dyes – TMRM for mitochondria, LysoTracker® Red for lysosomes and Hoechst 33342 for nuclei. Results: Under the normal conditions, the green fluorescence of DSSQ1 was localized to the cytosol, lysosomes, nuclear membrane and within mitochondria. Oxidative stress (extracellular H2O2, 100 μM) significantly decreased the loading efficiency of the redox sensor DSSQ1 into the fibroblasts, while reducing agent (extracellular N-acetyl cysteine, 10 mM), which is known to increase intracellular levels of glutathione and cytoplasmic redox state, enhanced the uptake of DSSQ1. Conclusion: The chemical structure of DSSQ1 allows permeability of compound without losing viability of cells. The compound is distributed within the cytoplasm, and localizes to lysosomes, mitochondria and nuclear membrane, but excluded from the nuclei. DSSQ1 accumulation is affected by redox status of cells and could be used to monitor the redox status of the cell