Reaction of rats organ cells to inhibition of protein biosynthesis by sublethal doses of cycloheximide

Time-dependent responses of cellular systems in rat organs and Fe(3+)-transferrin and Cu(2+)-ceruloplasmin pools in blood to the blocking of translation by sublethal doses of cycloheximide (CHI) was studied by EPR spectroscopy and radioisotope techniques. It was shown that, within the early post-CHI-treatment time, the suppression of deoxyribonucleotide and DNA biosynthesis, the activation of catabolic enzymes, the inhibition of electron transfer in the mitochondrial electron transport chain, the activation and the following inactivation of cytochrome P-450, and an intensive production of nitrosyl complexes in rat blood and organs occur. In addition, the activation of the synthesis of steroid hormones in adrenal gland was revealed within 1-24 h after cycloheximide injection. In response to these metabolic disturbances, nonspecific compensatory recovery reactions developed, first of all, the "reprograming" of the translation process to produce new protein-synthesizing elements instead of cycloheximide-blocked ones. The activation of protein synthesis promotes the recovery of deoxyribonucleotide and DNA synthesis, the restoration of the redox state of mitochondrial and microsomal electron transport chains in organs as well as an increase of Fe(3+)-transferrin and Cu(2+)-ceruloplasmin pools in rat blood. These metabolic processes result in the full recovery of the functional ability of organs.     

The effect of gamma-hydroxybutyric acid on the biosynthesis of macromolecules and metabolic paramagnetic centers in hepatocytes in acute radiation injury

It has been shown, that the single injection of gamma-hydroxybutyric acid (GHBA) (100 mg/kg) to mice 30 min before irradiation (6 Gy) prevents whole-body irradiation-induced inhibition of DNA at early post-irradiation period. GHBA stimulates the biosynthesis of macromolecules at 1-2 days after irradiation. GHBA also prevents the increase in the degree of reduction of the mitochondrial and microsomal electron transport chains at early post-irradiation time (up to 2 h.), that takes place only under irradiation. It means, that GHBA inhibits the production of O2 radicals, which induce lipid peroxidation processes at post-irradiation period.     

Increased biosynthesis of pyruvate kinase under hypoxic conditions in mammalian cells

The rate of biosynthesis of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) was compared in cells maintained under normoxic or hypoxic conditions. L8 cells (a myoblast cell line) were pulse-labeled with [3H]leucine and incorporation of radioactivity into pyruvate kinase was measured after quantitative affinity separation with anti-pyruvate kinase monoclonal antibody. During chronic hypoxia there is an increased rate of biosynthesis of pyruvate kinase leading to an increase in enzyme content and augmented glycolytic capacity. An inhibitor of the electron transport chain, antimycin A, was used to determine whether changes in pyruvate kinase content occurring during hypoxia are a result of reduction in molecular oxygen directly or an indirect consequence of oxygen depletion. Pyruvate kinase activity increased during chronic antimycin A exposure under normoxic conditions. The increase was quantitatively accounted for by an increase in cellular pyruvate kinase enzyme content. This suggested that decreases in the levels of molecular O2 are not the direct stimulus for the increased content of pyruvate kinase. It is more likely that the increased pyruvate kinase content results from depressed rates of electron transport through the mitochondrial electron transport chain.     

Redox properties of electron-transfer flavoprotein ubiquinone oxidoreductase as determined by EPR-spectroelectrochemistry.

We have determined the formal potential values for each electron transfer to electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), in order to further characterize the thermodynamics of electron transport from various acyl-CoA thioesters to the mitochondrial ubiquinone pool. ETF-QO contains one [4Fe-4S]2+,1+ cluster and one FAD prosthetic group. A preliminary visible-spectroelectrochemical titration showed that the two redox centers were reduced almost simultaneously. Since the visible spectra of the chromophores overlap, it was not possible to resolve the formal potential value for each electron transfer to the protein using this method. Accordingly, an EPR-spectroelectrochemical cell was designed so that each formal potential value could be resolved by EPR quantitation of the flavin semiquinone and the reduced iron-sulfur cluster during the titration. The formal potential values for electron transfer to ETF-ubiquinone oxidoreductase at pH 7.5 and 4 degrees C were E1 degrees' = +0.028 V and E2 degrees' = -0.006 V for the first and second electron transfers, respectively, to the FAD and E degrees' = +0.047 V for the iron-sulfur cluster. The thermodynamics of electron transport from the acyl-CoA substrates of beta-oxidation to the mitochondrial electron transport chain have been fully resolved with completion of this work. The results are discussed in terms of their significance to the overall electron transport process from beta-oxidation.     

Inhibition of complex I by Ca2+ reduces electron transport activity and the rate of superoxide anion production in cardiac submitochondrial particles

Declines in the rate of mitochondrial electron transport and subsequent increases in the half-life of reduced components of the electron transport chain can stimulate O2*- formation. We have previously shown that, in solubilized cardiac mitochondria, Ca2+ mediates reversible free radical-induced inhibition of complex I. In the study presented here, submitochondrial particles prepared from rat heart were utilized to determine the effects of Ca2+ on specific components of the respiratory chain and on the rates of electron transport and O2*- production. The results indicate that complex I is inactivated when submitochondrial particles are treated with Ca2+. Inactivation was specific to complex I with no alterations in the activities of other electron transport chain complexes. Complex I inactivation by Ca2+ resulted in the reduction of NADH-supported electron transport activity. In contrast to the majority of electron transport chain inhibitors, Ca2+ suppressed the rate of O2*- production. In addition, while inhibition of complex III stimulated O2*- production, Ca2+ reduced the relative rate of O2*- production, consistent with the magnitude of complex I inhibition. Evidence indicates that complex I is the primary source of O2*- released from this preparation of submitochondrial particles. Ca2+ therefore inhibits electron transport upstream of site(s) of free radical production. This may represent a means of limiting O2*- production by a compromised electron transport chain.     

Electron-spin resonance studies of the bound iron-sulfur centers in Photosystem I. II. Correlation of P-700 triplet production with urea/ferricyanide inactivation of the iron-sulfur clusters

Photosystem I charge separation in a subchloroplast particle isolated from spinach was investigated by electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur centers by urea-ferricyanide treatment. Previous work demonstrated a differential decrease in iron-sulfur centers A, B and X which indicated that center X serves as a branch point for parallel electron flow through centers A and B (Golbeck, J.H. and Warden, J.T. (1982) Biochim. Biophys. Acta 681, 77-84). We now show that during inactivation the disappearance of iron-sulfur centers A, B, and X correlates with the appearance of a spin-polarized triplet ESR signal with [D] = 279 X 10(-4) cm-1 and [E] = 39 X 10(-4) cm-1. The triplet resonances titrate with a midpoint potential of +380 +/- 10 mV. Illumination of the inactivated particles results in the generation of an asymmetric ESR signal with g = 2.0031 and delta Hpp = 1.0 mT. Deconvolution of the P-700+ contribution to this composite resonance reveals the spectrum of the putative primary acceptor species A0, which is characterized by g = 2.0033 +/- 0.0004 and delta Hpp = 1.0 +/- 0.2 mT. The data presented in this report do not substantiate the participation of the electron acceptor A1 in PS I electron transport, following destruction of the iron-sulfur cluster corresponding to center X. We suggest that A1 is closely associated with center X and that this component is decoupled from the electron-transport path upon destruction of center X. The inability to photoreduce A1 in reaction centers lacking a functional center X may result from alteration of the reaction center tertiary structure by the urea-ferricyanide treatment or from displacement of A1 from its binding site.     

Effect of sodium nitroprusside on H+-ATPase activity and ATP concentration in Candida albicans

ATP hydrolysis by plasma membrane H+-ATPase from Candida albicans has been investigated in presence of nitric oxide and various nutrients (sugars and amino acids). Sodium nitroprusside (SNP) was used as nitric oxide donor. It was found that ATP concentration decreased in SNP treated cells which was more in presence of sugars like glucose, xylose and 2-deoxy-D-glucose and amino acids as compared to their respective controls. The activity of H+-ATPase from plasma membrane decreased by 70 % in SNP treated cells. Both in vivo and in vitro treatments of SNP showed almost similar effects of decrease in ATPase activity. Effect of SNP was more pronounced in presence of nutrients. Interestingly, it was observed that vanadate did not show any independent effect in presence of nitric oxide. Several workers have reported similar type of results with other P-type ATPases. For the first time, it was observed in the present study that in presence of nitric oxide, H+-ATPase activity decreased like other P-type ATPases. Our study indicated that NO had a significant effect on ATP synthesis and activity of H+- ATPase. In the presence of NO, the ATP concentration was decreased indicating it affected mitochondrial electron transport chain. It may be concluded that NO, not only affects (inhibit) mitochondrial electron transport chain but also interferes with H+- ATPase of plasma membrane by changing its conformation resulting in decreased activity.     

A mechanism of sulfite neurotoxicity: direct inhibition of glutamate dehydrogenase

Exposure of Neuro-2a and PC12 cells to micromolar concentrations of sulfite caused an increase in reactive oxygen species and a decrease in ATP. Likewise, the biosynthesis of ATP in intact rat brain mitochondria from the oxidation of glutamate was inhibited by micromolar sulfite. Glutamate-driven respiration increased the mitochondrial membrane potential (MMP), and this was abolished by sulfite but the MMP generated by oxidation of malate and succinate was not affected. The increased rate of production of NADH from exogenous NAD+ and glutamate added to rat brain mitochondrial extracts was inhibited by sulfite, and mitochondria preincubated with sulfite failed to reduce NAD+. Glutamate dehydrogenase (GDH) in rat brain mitochondrial extract was inhibited dose-dependently by sulfite as was the activity of a purified enzyme. An increase in the Km (glutamate) and a decrease in Vmax resulting in an attenuation in Vmax/Km (glutamate) at 100 microm sulfite suggest a mixed type of inhibition. However, uncompetitive inhibition was noted with decreases in both Km (NAD+) and Vmax, whereas Vmax/Km (NAD+) remained relatively constant. We propose that GDH is one target of action of sulfite, leading to a decrease in alpha-ketoglutarate and a diminished flux through the tricarboxylic acid cycle accompanied by a decrease in NADH through the mitochondrial electron transport chain, a decreased MMP, and a decrease in ATP synthesis. Because glutamate is a major metabolite in the brain, inhibition of GDH by sulfite could contribute to the severe phenotype of sulfite oxidase deficiency in human infants.     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca2+ transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca2+ transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mCICR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mCICR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mCICR and PTP opening. mCICR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca2+ transport and PTP opening are largely dependent on electron transport and energy coupling.     

Age-associated damage in mitochondrial DNA in human hearts

Damage to mitochondrial DNA seems to be involved in the etiology of age-associated degenerative diseases. The aim of this study is to elucidate effects of aging on human mitochondrial DNA. 8-Hydroxy-deoxyguanosine, a product of free radical damage to deoxyguanosine, is reported to cause random point mutations. In human mitochondrial DNA, 8-hydroxy-deoxyguanosine increased exponentially with age, and the population of mitochondrial DNA with deletion increased also exponentially with age. Furthermore, a clear correlation existed between the accumulation of 8-hydroxy-deoxyguanosine and that of mitochondrial DNA with deletion. We also determined the effects of aging on rat mitochondrial function together with 8-hydroxy-deoxyguanosine content in mitochondrial DNA. The activities of complexes I and IV of the mitochondrial electron transport chain decreased significantly in rats aged 100 weeks compared with those in rats aged 7 weeks. A concomitant increase in 8-hydroxy-deoxyguanosine was observed in mitochondrial DNA of rats aged 100 weeks. From our results, it is concluded that the age-associated accumulation of somatically acquired oxygen damage together with deletions in mitochondrial DNA might be important contributors to the deterioration of cardiac function associated with age.Abbreviations mtDNA mitochondrial DNA - 8-OH-dG 8-Hydroxy-Deoxyguanosine - dG Deoxyguanosine - HPLC/MS Micro-High Performance Liquid Chromatography/Mass Spectrometry     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca²⁺ transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca²⁺ transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mCICR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mCICR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mCICR and PTP opening. mCICR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca²⁺ transport and PTP opening are largely dependent on electron transport and energy coupling.     

Mitochondrial free radical generation, oxidative stress, and aging

Mitochondria have been described as "the powerhouses of the cell" because they link the energy-releasing activities of electron transport and proton pumping with the energy conserving process of oxidative phosphorylation, to harness the value of foods in the form of ATP. Such energetic processes are not without dangers, however, and the electron transport chain has proved to be somewhat "leaky." Such side reactions of the mitochondrial electron transport chain with molecular oxygen directly generate the superoxide anion radical (O2*-), which dismutates to form hydrogen peroxide (H2O2), which can further react to form the hydroxyl radical (HO*). In addition to these toxic electron transport chain reactions of the inner mitochondrial membrane, the mitochondrial outer membrane enzyme monoamine oxidase catalyzes the oxidative deamination of biogenic amines and is a quantitatively large source of H2O2 that contributes to an increase in the steady state concentrations of reactive species within both the mitochondrial matrix and cytosol. In this article we review the mitochondrial rates of production and steady state levels of these reactive oxygen species. Reactive oxygen species generated by mitochondria, or from other sites within or outside the cell, cause damage to mitochondrial components and initiate degradative processes. Such toxic reactions contribute significantly to the aging process and form the central dogma of "The Free Radical Theory of Aging." In this article we review current understandings of mitochondrial DNA, RNA, and protein modifications by oxidative stress and the enzymatic removal of oxidatively damaged products by nucleases and proteases. The possible contributions of mitochondrial oxidative polynucleotide and protein turnover to apoptosis and aging are explored.     

Regulation of the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels, Citellus undulatus

1. The rates of oxidation of various substrates (beta-hydroxybutyrate, succinate, ascorbate + TMPD) and the rate of ATP synthesis in liver mitochondria from active and hibernating ground squirrels were measured. 2. It was shown that the rate of mitochondrial respiration is significantly lower in hibernating animals than in active animals. 3. The degree of inhibition of mitochondrial respiration in hibernating ground squirrels was found to correlate with the length of the respiratory chain fragment involved in the oxidation of a given substrate. 4. The inhibition of mitochondrial respiration in hibernating animals was accompanied by a decrease in the rate of ATP synthesis. 5. The activity of phospholipase A2 in liver mitochondria from hibernating ground squirrels was found to be decreased. The activation of phospholipase A2 by Ca2+ ions eliminated the inhibition of respiration almost completely. 6. It was assumed that the inhibition of mitochondrial respiration during hibernation is (a) related to the suppression of phospholipase A2 activity and (b) caused by the reduced rates of electron transport through the respiratory chain and/or of substrate transport across the mitochondrial membrane.     

Effect of ionizing radiation on the superoxide dismutase content and turnover in the rat liver

A study was made of the effect of ionizing radiation on the activity, concentration, biosynthesis, transport and disintegration of cytosol and mitochondrial forms of superoxide dismutase in rat hepatocytes. It was shown that after total-body X-irradiation there was a tendency toward a decrease in the activity of the enzyme while its concentration in the mitochondrial fraction increased. Stimulation of the processes of biosynthesis, transport and disintegration of superoxide dismutase in both fractions was also detected. The data obtained are discussed with regard to the development of radiation sickness.     

Assessment of the role of oxygen and mitochondria in heat shock induction of radiation and thermal resistance in Saccharomyces cerevisiae

In response to a heat shock, the yeast Saccharomyces cerevisiae undergoes a large increase in its resistance to heat and, by the induction of its recombinational DNA repair capacity, a corresponding increase in resistance to radiation. Yeast which lack mitochondrial DNA, mitochondria-controlled protein synthetic apparatus, aerobic respiration, and electron transport rho 0 strain) were used to assess the role of O2, mitochondria, and oxidative processes controlled by mitochondria in the induction of these resistances. We have found that rho 0 yeast grown and heat shocked in either the presence or absence of O2 are capable of developing both radiation and heat resistance. We conclude that neither the stress signal nor its cellular consequences of induced heat and radiation resistance are directly dependent on O2, mitochondrial DNA, or mitochondria-controlled protein synthetic or oxidative processes.     

Deletion of Von Willebrand A Domain Containing Protein (VWA8) raises activity of mitochondrial electron transport chain complexes in hepatocytes

VWA8 (Von Willebrand A Domain Containing Protein 8) is a AAA+ ATPase that is localized to the mitochondrial matrix and is widely expressed in highly energetic tissues. Originally found to be higher in abundance in livers of mice fed a high fat diet, deletion of the VWA8 gene in differentiated mouse AML12 hepatocytes unexpectedly produced a phenotype of higher mitochondrial and nonmitochondrial oxidative metabolism, higher ROS (reactive oxygen species) production mainly from NADPH oxidases, and increased HNF4a expression. The purposes of this study were first, to determine whether higher mitochondrial oxidative capacity in VWA8 null hepatocytes is the product of higher capacity in all aspects of the electron transport chain and oxidative phosphorylation, and second, the density of cristae in mitochondria and mitochondrial content was measured to determine if higher mitochondrial oxidative capacity is accompanied by greater cristae area and mitochondrial abundance. Electron transport chain complexes I, II, III, and IV activities all were higher in hepatocytes in which the VWA8 gene had been deleted using CRISPR/Cas9. A comparison of abundance of proteins in electron transport chain complexes I, III and ATP synthase previously determined using an unbiased proteomics approach in hepatocytes in which VWA8 had been deleted showed agreement with the activity assays. Mitochondrial cristae, the site where electron transport chain complexes are located, were quantified using electron microscopy and stereology. Cristae density, per mitochondrial area, was almost two-fold higher in the VWA8 null cells (P < 0.01), and mitochondrial area was two-fold higher in the VWA8 null cells (P < 0.05). The results of this study allow us to conclude that despite sustained, higher ROS production in VWA8 null cells, a global mitochondrial compensatory response was maintained, resulting in overall higher mitochondrial oxidative capacity.