Segregational Respiratory-Deficient Mutants of a “Petite Negative” Yeast Schizosaccharomyces pombe 972h−

No viable respiratory-deficient mutants of Schizosaccharomyces pombe 972h(-) could be obtained by acriflavine and ethidium bromide treatments. These mutagens induce 15 to 70% of microcolonies which, after a growth-lag of a few days, further develop into normal, respiratory-competent colonies. These results suggest that unstable petites were induced. Segregational respiratory-deficient mutants resistant to cobalt sulfate inhibition were isolated. Some of these strains are deficient in cytochrome a + a(3) and respire at low rates. The morphology of their mitochondrial membranes is modified: either the cristae are absent or they show aberrant concentric or tubular structures. Segregational mutants resistant to the respiratory inhibitors, 2,4-dinitrophenol or decamethylene diguanidine, were obtained. Neither mitochondrial structure nor function seems to be modified in these mutants. A segregational mutant resistant to benzimidazole inhibition does not grow on glycerol, although neither growth on glucose nor respiration appear to be affected.     

Effect of palmitoylcarnitine on mitochondrial activities

Mitochondria from pea (Pisum sativum L.) cotyledons and potato (Solanum tuberosum L.) tubers exhibited a palmitoyl carnitine-dependent, KCN-sensitive stimulation of the oxygen uptake measured in the presence of 0.2mmol·^–1 malate (sparker malate), provided a certain concentration range of palmitoylcarnitine was observed. Above this concentration range, which was dependent on the bovine serum albumin (BSA) concentration of the reaction mixture, the mitochondrial oxygen uptake was inhibited by palmitoylcarnitine. Palmitoylcarnitine (racemate) and palmitoyl-l-carnitine were equally effective in stimulating/inhibiting mitochondrial oxygen uptake in the presence of sparker malate. The mitochondrial membrane potential generated in the presence of sparker malate was partially dissipated by palmitoyl-lcarnitine concentrations stimulating the mitochondrial oxygen uptake. The formation of acid-soluble radioactivity in reaction mixtures provided with [1-¹⁴C]palmitoyll-carnitine was considerably lower than that expected minimally if the palmitoyl-l-carnitine-stimulated oxygen uptake resulted from palmitoyl-l-carnitine oxidation sparked by malate. Palmitoylcarnitine concentrations resulting in stimulation of the mitochondrial oxygen uptake in the presence of sparker malate also led to a stimulation of succinate-cytochrome c reductase activity, as well as to an increase in the measurable activities of mitochondrial matrix enzymes, indicating loss of both mitochondrial integrity and mitochondrial enzyme latency in the presence of palmitoylcarnitine. Correspondingly, malate-dependent NADH formation was stimulated by palmitoylcarnitine. Neither NAD reduction nor oxygen uptake were observed when the mitochondria were provided with palmitoylcarnitine only. The oxygen uptake due to glycine oxidation by mitochondria from green sunflower (Helianthus annuus L.) cotyledons was affected by palmitoylcarnitine in a similar manner to the oxygen uptake of pea cotyledon and potato tuber mitochondria in the presence of sparker malate. The results lead to the conclusion that the palmitoylcarnitine-dependent stimulation of mitochondrial oxygen uptake observed in the presence of sparker malate results substantially from an enhanced malate oxidation due to the detergent effect of palmitoylcarnitine on the mitochondrial membranes, rather than from palmitoylcarnitine -oxidation.Abbreviations BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophyenylhydrazone The work was supported by the Deutsche Forschungsgemeinschaft.     

Anisotropic action of cetyl pyridinium chloride on rat heart mitochondria

This work reports experiments that show that in rat heart mitochondria, the alkyl cation cetyl pyridinium chloride induces inhibition of the electron transport with NAD-dependent substrates. It also induces an enhancement of oxygen uptake with succinate as substrate, stimulation of adenosine triphosphatase activity, release of Ca²⁺ that have been accumulated, and inhibition of the energy-dependent uptake of ethidium bromide; these findings suggest that cetyl pyridinium chloride induces a collapse of membrane potential. The experiments carried out with submitochondrial particles showed that this reagent inhibits the oxidation of NADH, provided an uncoupler is added to the system. According to these data it is proposed that the latter effect is due to the binding of cetyl pyridinium chloride to the inner mitochondrial membrane in a site that faces the cytosol.     

Uncoupling protein 2: a novel player in neuroprotection

A recent report provides exciting new evidence that suggests that uncoupling protein 2 (UCP2), a mitochondrial protein expressed in specific cells of numerous tissues, might be neuroprotective by reducing mitochondrial Ca²⁺ uptake and preventing mitochondrial accumulation of reactive oxygen species (ROS) following cerebral ischemia. The mitochondrial sequestration of Ca²⁺ and ROS, which depends on the mitochondrial membrane potential (ΔΨ_m), is a deleterious consequence of excitotoxicity. A neuroprotective role for Ucp2 is consistent with the already proposed property of this gene in mitigating cellular damage caused by ROS.     

Iron transport from sepharose-bound transferrin

To ascertain whether transferrin need enter the reticulocyte to deliver its iron after the association of transferrin with the cell membrane, {¹²⁵I, ⁵⁹Fe-}labeled transferrin was covalently bound to Sepharose beads. Iron uptake from Sepharose-bound transferrin into rabbit reticulocytes was about 9% that from free transferrin while heme synthesis was more efficient at nearly 19%. Similar results were obtained with murine transferrin and murine reticulocytes.These results indicate that the entrance of transferrin inside the cell is not an obligatory step in the process of iron uptake in rabbit and murine reticulocytes.     

Alteration in mitochondrial Ca<Superscript>2+</Superscript> uptake disrupts insulin signaling in hypertrophic cardiomyocytes

Background

Cardiac hypertrophy is characterized by alterations in both cardiac bioenergetics and insulin sensitivity. Insulin promotes glucose uptake by cardiomyocytes and its use as a substrate for glycolysis and mitochondrial oxidation in order to maintain the high cardiac energy demands. Insulin stimulates Ca²⁺ release from the endoplasmic reticulum, however, how this translates to changes in mitochondrial metabolism in either healthy or hypertrophic cardiomyocytes is not fully understood.

Results

In the present study we investigated insulin-dependent mitochondrial Ca²⁺ signaling in normal and norepinephrine or insulin like growth factor-1-induced hypertrophic cardiomyocytes. Using mitochondrion-selective Ca²⁺-fluorescent probes we showed that insulin increases mitochondrial Ca²⁺ levels. This signal was inhibited by the pharmacological blockade of either the inositol 1,4,5-triphosphate receptor or the mitochondrial Ca²⁺ uniporter, as well as by siRNA-dependent mitochondrial Ca²⁺ uniporter knockdown. Norepinephrine-stimulated cardiomyocytes showed a significant decrease in endoplasmic reticulum-mitochondrial contacts compared to either control or insulin like growth factor-1-stimulated cells. This resulted in a reduction in mitochondrial Ca²⁺ uptake, Akt activation, glucose uptake and oxygen consumption in response to insulin. Blocking mitochondrial Ca²⁺ uptake was sufficient to mimic the effect of norepinephrine-induced cardiomyocyte hypertrophy on insulin signaling.

Conclusions

Mitochondrial Ca²⁺ uptake is a key event in insulin signaling and metabolism in cardiomyocytes.

     

Mitochondrial ATP-Sensitive K<Superscript>+</Superscript> Channels Prevent Oxidative Stress,Permeability Transition and Cell Death

Ischemia followed by reperfusion results in impairment of cellular and mitochondrial functionality due to opening of mitochondrial permeability transition pores. On the other hand, activation of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) protects the heart against ischemic damage. This study examined the effects of mitoK_ATP and mitochondrial permeability transition on isolated rat heart mitochondria and cardiac cells submitted to simulated ischemia and reperfusion (cyanide/aglycemia). Both mitoK_ATP opening, using diazoxide, and the prevention of mitochondrial permeability transition, using cyclosporin A, protected against cellular damage, without additive effects. MitoK_ATP opening in isolated rat heart mitochondria slightly decreased Ca²⁺ uptake and prevented mitochondrial reactive oxygen species production, most notably in the presence of added Ca²⁺. In ischemic cells, diazoxide decreased ROS generation during cyanide/aglycemia while cyclosporin A prevented oxidative stress only during simulated reperfusion. Collectively, these studies indicate that opening mitoK_ATP prevents cellular death under conditions of ischemia/reperfusion by decreasing mitochondrial reactive oxygen species release secondary to Ca²⁺ uptake, inhibiting mitochondrial permeability transition.     

Dimers of nicotinamide adenine dinucleotide: New evidence for the structure and the involvement in an enzymatic redox process

The composition and the structure of the product from the known electrochemical dimerization of the NAD⁺ have been conclusively demonstrated. A detailed analysis of the ¹H and ¹³C nmr spectra has in fact led to the conclusion that the product contains three diastereoisomeric dimers of the 4,4′-tetrahydrobipyridyl type. Furthermore, the cytoplasmic fraction obtained from a standard mitochondrial preparation of rat liver has been shown to catalyze the oxygen uptake by the dimers. A 1 : 1 molar ratio of the reagents in the redox process is indicated by manometric data on oxygen uptake complemented by spectrophotometric analysis of the oxidized substrates, suggesting that H₂O₂ is the reduction product. NAD⁺ was identified as the oxidation product by an enzymatic method.     

Spegazzinine,a new inhibitor of mitochondrial oxidative phosphorylation

The effects of spegazzinine, a dihydroindole alkaloid, on mitochondrial oxidative phosphorylation were studied.Spegazzinine inhibited coupled respiration and phosphorylation in rat liver mitochondria. The I₅₀ was 120 μM. Uncouplers released the inhibition of coupled respiration. Arsenate-stimulated mitochondrial respiration was partially inhibited by spegazzinine. The stimulation of mitochondrial respiration by Ca²⁺ and the proton ejection associated with the ATP-dependent Ca²⁺ uptake were not affected by the alkaloid.Oxidative phosphorylation and the P_i-ATP exchange reaction of phosphorylating beef heart submitochondrial particles were strongly inhibited by spegazzinine (I₅₀, 50 μM) while the ATP-dependent reactions, reduction of NAD⁺ by succinate and the pyridine nucleotides transhydrogenase were less sensitive (I₅₀, 125 μM). Oxygen uptake by submitochondrial particles was not affected.The 2,4-dinitrophenol-stimulated ATPase activity of rat liver mitochondria was not affected by 300 μM spegazzinine, a concentration of alkaloid that completely inhibited phosphorylation. However, higher concentrations of spegazzinine did partially inhibit it. The ATPase activities of submitochondrial particles, insoluble and soluble ATPases were also partially inhibited by high concentrations of spegazzinine.The inhibitory properties of spegazzinine on energy transfer reactions are compared with those of oligomycin, aurovertin and dicyclohexylcarbodiimide. It is concluded that spegazzinine effects are very similar to the effects of aurovertin and that its site of action may be the same or near the site of aurovertin.     

The physical and chemical properties of a chicken egg white glycoprotein purified by nondenaturing methodology

The oxidation of ethanol by the liver produces acetaldehyde, which is a highly reactive compound. Low concentrations of acetaldehyde inhibited mitochondrial respiration with glutamate, β-hydroxybutyrate, or α-ketoglutarate as substrates, but not with succinate or ascorbate. High concentrations led to respiratory inhibition with all substrates. Inhibition of succinate- and ascorbate-linked oxidation by acetaldehyde correlates with the inhibition of the activities of succinic dehydrogenase and cytochrome oxidase. A site more sensitive to acetaldehyde appears to be localized prior to the NADH-ubiquinone oxidoreductase segment of the respiratory chain. Acetaldehyde inhibits energy production by the mitochondria, as evidenced by its inhibition of respiratory control, oxidative phosphorylation, the rate of phosphorylation, and the ATP-³²P exchange reaction. Energy utilization is also inhibited, in view of the decrease in both substrate- and ATP-supported Ca²⁺ uptake, and the reduction in Ca²⁺-stimulated oxygen uptake and ATPase activity. The malate-aspartate, α-glycerophosphate, and fatty acid shuttles for the transfer of reducing equivalents, and oxidation by mitochondria, were highly sensitive to acetaldehyde. Acetaldehyde also inhibited the uptake of anions which participate in the shuttles. The inhibition of the shuttles is apparently caused by interference with NAD⁺-dependent state 3 respiration and anion entry and efflux. Ethanol (6–80 mm) had no significant effect on oxygen consumption, anion uptake, or mitochondrial energy production and utilization. The data suggest that acetaldehyde may be implicated in some of the toxic effects caused by chronic ethanol consumption.     

Microaerobic respiration and oxidative phosphorylation by soybean nodule mitochondria: implications for nitrogen fixation

The infected cells of soybean (Glycine max) root nodules require ATP production for ammonia assimilation and purine synthesis under microaerobic conditions. It is likely that the bulk of this demand is supplied through mitochondrial oxidative phosphorylation. Mitochondria purified from root nodules respired and synthesized ATP in sub-micromolar oxygen concentrations as measured by leghaemoglobin spectroscopy and luciferase luminescence. Both oxygen uptake and the apparent ATP/O ratio declined significantly as the oxygen concentration fell below 100 μmol m^?3. Cytochrome-pathway respiration by root nodule mitochondria had a higher apparent affinity for oxygen (Km 50 μmol m^?3) than did mitochondria isolated from roots (Km 125 μmol m^?3). Electron micrographs showed that mitochondria predominated at the periphery of infected cells adjacent to gas-filled intercellular spaces, where the oxygen concentration is predicted to be highest. Calculations of oxygen concentration and nitrogen fixation rates on an infected cell basis suggest that the measured rates of ATP production by isolated mitochondria are sufficient for the quantifiable in vivo requirements of ammonia assimilation and purine synthesis. The possible roles of mitochondrial respiration in the control of infected cell metabolism are also discussed.     

Effect of clofibrate on the adenosine triphosphatase activity of rat liver mitochondria.

The antihypercholesterolemic drug clofibrate (ethyl-α-p-chlorophenoxyisobutyrate) stimulated the latent ATPase activity and “superstimulated” the uncoupler-induced ATPase activity of rat-liver mitochondria. Addition of clofibrate decreased the turbidity of mitochondrial suspensions and released considerable amount of mitochondrial protein into solution. In these properties it closely resembled detergents like Triton X-100 and deoxycholate. However, unlike the detergents, clofibrate required the presence of a permeant cation for its disruptive action. Also, it was without any such effect on sonic submitochondrial particles. The drug enhanced the uptake of both Mg² and Cl^? by mitochondria suggesting that osmotic swelling precedes lysis. Sonic submitochondrial particles prepared in the presence of clofibrate showed a greater yield and comparable ATPase activity.     

Mitochondrial Calcium Uniporter Activity Is Dispensable for MDA-MB-231 Breast Carcinoma Cell Survival

Calcium uptake through the mitochondrial Ca²⁺ uniporter (MCU) is thought to be essential in regulating cellular signaling events, energy status, and survival. Functional dissection of the uniporter is now possible through the recent identification of the genes encoding for MCU protein complex subunits. Cancer cells exhibit many aspects of mitochondrial dysfunction associated with altered mitochondrial Ca²⁺ levels including resistance to apoptosis, increased reactive oxygen species production and decreased oxidative metabolism. We used a publically available database to determine that breast cancer patient outcomes negatively correlated with increased MCU Ca²⁺ conducting pore subunit expression and decreased MICU1 regulatory subunit expression. We hypothesized breast cancer cells may therefore be sensitive to MCU channel manipulation. We used the widely studied MDA-MB-231 breast cancer cell line to investigate whether disruption or increased activation of mitochondrial Ca²⁺ uptake with specific siRNAs and adenoviral overexpression constructs would sensitize these cells to therapy-related stress. MDA-MB-231 cells were found to contain functional MCU channels that readily respond to cellular stimulation and elicit robust AMPK phosphorylation responses to nutrient withdrawal. Surprisingly, knockdown of MCU or MICU1 did not affect reactive oxygen species production or cause significant effects on clonogenic cell survival of MDA-MB-231 cells exposed to irradiation, chemotherapeutic agents, or nutrient deprivation. Overexpression of wild type or a dominant negative mutant MCU did not affect basal cloning efficiency or ceramide-induced cell killing. In contrast, non-cancerous breast epithelial HMEC cells showed reduced survival after MCU or MICU1 knockdown. These results support the conclusion that MDA-MB-231 breast cancer cells do not rely on MCU or MICU1 activity for survival in contrast to previous findings in cells derived from cervical, colon, and prostate cancers and suggest that not all carcinomas will be sensitive to therapies targeting mitochondrial Ca²⁺ uptake mechanisms.     

Inhibition of the mitochondrial calcium uniporter prevents IL-13 and allergen-mediated airway epithelial apoptosis and loss of barrier function

Mitochondria are increasingly recognized as key mediators of acute cellular stress responses in asthma. However, the distinct roles of regulators of mitochondrial physiology on allergic asthma phenotypes are currently unknown. The mitochondrial Ca²⁺ uniporter (MCU) resides in the inner mitochondrial membrane and controls mitochondrial Ca²⁺ uptake into the mitochondrial matrix. To understand the function of MCU in models of allergic asthma, in vitro and in vivo studies were performed using models of functional deficiency or knockout of MCU. In primary human respiratory epithelial cells, MCU inhibition abrogated mitochondrial Ca²⁺ uptake and reactive oxygen species (ROS) production, preserved the mitochondrial membrane potential and protected from apoptosis in response to the pleiotropic Th2 cytokine IL-13. Consequently, epithelial barrier function was maintained with MCU inhibition. Similarly, the endothelial barrier was preserved in respiratory epithelium isolated from MCU-/- mice after exposure to IL-13. In the ovalbumin-model of allergic airway disease, MCU deficiency resulted in decreased apoptosis within the large airway epithelial cells. Concordantly, expression of the tight junction protein ZO-1 was preserved, indicative of maintenance of epithelial barrier function. These data implicate mitochondrial Ca²⁺ uptake through MCU as a key controller of epithelial cell viability in acute allergic asthma.     

TRPM2 Channels Protect against Cardiac Ischemia-Reperfusion Injury: ROLE OF MITOCHONDRIA*

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of G_Ca to G_Na of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca²⁺ uptake, ATP levels, and O₂ consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca²⁺ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca²⁺ uptake was likely due to both lower ψ_m and MCU activity. Similar to isolated myocytes, O₂ consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.     

Mitochondria from rat uterine smooth muscle possess ATP-sensitive potassium channel

The objective of this study was to detect ATP-sensitive K⁺ uptake in rat uterine smooth muscle mitochondria and to determine possible effects of its activation on mitochondrial physiology. By means of fluorescent technique with usage of K⁺-sensitive fluorescent probe PBFI (potassium-binding benzofuran isophthalate) we showed that accumulation of K ions in isolated mitochondria from rat myometrium is sensitive to effectors of K_ATP-channel (ATP-sensitive K⁺-channel) – ATP, diazoxide, glibenclamide and 5HD (5-hydroxydecanoate). Our data demonstrates that K⁺ uptake in isolated myometrium mitochondria results in a slight decrease in membrane potential, enhancement of generation of ROS (reactive oxygen species) and mitochondrial swelling. Particularly, the addition of ATP into incubation medium led to a decrease in mitochondrial swelling and ROS production, and an increase in membrane potential. These effects were eliminated by diazoxide. If blockers of K_ATP-channel were added along with diazoxide, the effects of diazoxide were removed. So, we postulate the existence of K_ATP-channels in rat uterus mitochondria and assume that their functioning may regulate physiological conditions of mitochondria, such as matrix volume, ROS generation and polarization of mitochondrial membrane.     

Oxygen consumption and the composition of skeletal muscle tissue after training and inactivation in the European woodmouse (Apodemus sylvaticus)

Summary In European woodmice the amount and intensity of daily activity was compared to oxygen uptake and to the potential for oxidative metabolism of heart and skeletal muscle. One group of animals was inactivated by exposition to light during night time; another group of animals was trained by enforced running on a treadmill. The oxidative potential of the muscle tissue was assessed by morphometry of capillaries and mitochondria. A novel sampling technique was used which allowed us to obtain morphological data related to single muscles, to muscle groups, and finally to whole body muscle mass.Reducing the spontaneous activity by ten fold had no effect on oxygen uptake nor on capillaries or mitochondria in locomotory muscles. Mitochondrial volume was reduced, however, in heart and diaphragm. Enforced running increased the weight specific maximal oxygen uptake significantly. It also increased the mitochondrial volume in heart and diaphragm as well as in M. tibialis anterior. Capillary densities were neither affected by training nor by inactivation. A significant correlation was found between the capillary density and the volume density of mitochondria in all muscles analysed morphometrically. For the whole skeletal muscle mass of a European woodmouse the inner mitochondrial membranes were estimated to cover 30 m². The oxygen consumption per unit time and per unit volume of muscle mitochondrion was found to be identical in all groups of animals (4.9 ml O₂ min^–1 cm^–3).Symbols S _v (im,m) surface area of inner mitochondrial membranes per unit mitochondrial volume - V _v (mt, f) volume density of mitochondria (mitochondrial volume per fiber volume) - V (mt) total mitochondrial volume - V (f) muscle volume - N _A (c, f) capillary density - (f) mean fiber cross-sectional area     

Effect of cholesterol content on some physical and functional properties of mitochondria isolated from adult rat liver,fetal liver,cholesterol-enriched liver and hepatomas AH-130, 3924A and 5123

The choleterol to phospholipid ratio in mitochodria from hepatomas AH-130, 3924A and 5123 is higher than in the particles isolated from adult or fetal rat livers. Nearly all the cholesterol of hepatoma mitochonda is located in membranes. As in liver mitochondria, in the particles isolated from hepatoma AH-130 there is more cholesterol in the outer than in the inner membrane.In mitochondria from cholesterol-enriched liver and hepatomas, there occur a decrease in extent of hypoosmotic and phosphate-induced sweeling and decrease of conformational linked energy states. The phenomenon is more marked in particles which exhibit higher cholesterol to phospholopid ratios. A statistically significant negative correlation exists between the cholesterol to phospholipid ratio and extent of volume or conformation changes. No significant modifications of these parameters were found in fetal liver mitochondria.Cholesterol content does not influence K⁺ uptake by cholesterol-enriched or hepatoma mitochondria. Nor does cholesterol content affect the respiratory increment related to this uptake. As a consequence of K⁺ uptake, total mitochodrial water exchangeable with tritiated water rises 20% while sucrose-impermeable water rises 42–48% in both adult rat liver and hepatoma AH-130 mitochondria. Absorbance changes linked to ion uptake do not correspond merely to variations in mitochobdrial water content. Water content. Water is apparently not influenced by the cholesterol to phospholipid ratio. However, the ratio is signifacantly correlated to both extent and initial rate of absorbance decrease of mitochondrial suspension during K⁺ uptake. The higher the ratio, the lower the extent and and initial rate of absorbance decrease.