Glucocorticoid and estrogen receptors are reduced in mitochondria of lung epithelial cells in asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GRα and ERβ in lung tissue. Allergic airway inflammation caused reduction in mtGRα, mtERβ, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GRα and ERβ in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.     

Multiple cytochrome deficiency and deteriorated mitochondrial polypeptide composition in fatal infantile mitochondrial myopathy and renal dysfunction

Mitochondria isolated from the skeletal muscle of an infant with mitochondrial myopathy and renal dysfunction were analyzed. Activities of NADH dehydrogenase, succinate dehydrogenase, ubiquinol-cytochrome c oxidoreductase, and cytochrome c oxidase were severely decreased. Cytochromes aa3 and b were not detected in patient mitochondria, and the cytochrome c+c1 content was 14% of control. Immunoblotting demonstrated that the amount of cytochrome c oxidase subunits were markedly decreased in patient mitochondria. The polypeptide profile of patient mitochondria was quite different from that of control mitochondria. These results suggest that deterioration of mitochondria in a severe case of mitochondrial myopathy involves not only cytochrome c oxidase but also other mitochondrial proteins.     

Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin

Reactive oxygen species (ROS) are considered a key factor in mitochondrial dysfunction associated with brain aging process. Mitochondrial respiration is an important source of ROS and hence a potential contributor to brain functional changes with aging. In this study, we examined the effect of aging on cytochrome c oxidase activity and other bioenergetic processes such as oxygen consumption, membrane potential and ROS production in rat brain mitochondria. We found a significant age-dependent decline in the cytochrome c oxidase activity which was associated with parallel changes in state 3 respiration, membrane potential and with an increase in H₂O₂ generation. The cytochrome aa₃ content was practically unchanged in mitochondria from young and aged animals. The age-dependent decline of cytochrome c oxidase activity could be restored, in situ, to the level of young animals, by exogenously added cardiolipin. In addition, exposure of brain mitochondria to peroxidized cardiolipin resulted in an inactivation of this enzyme complex. It is suggested that oxidation/depletion of cardiolipin could be responsible, at least in part, for the decline of cytochrome c oxidase and mitochondrial dysfunction in brain aging. Melatonin treatment of old animals largely prevented the age-associated alterations of mitochondrial bioenergetic parameters. These results may prove useful in elucidating the molecular mechanisms underlying mitochondrial dysfunction associated with brain aging process, and may have implications in etiopathology of age-associated neurodegenerative disorders and in the development of potential treatment strategies.     

Cisplatin nephrotoxicity involves mitochondrial injury with impaired tubular mitochondrial enzyme activity

Cisplatin is a widely used antineoplastic agent. However, its major limitation is dose-dependent nephrotoxicity whose precise mechanism is poorly understood. Recent studies have suggested that mitochondrial dysfunction in tubular epithelium contributes to cisplatin-induced nephrotoxicity. Here the authors extend those findings by describing the role of an important electron transport chain enzyme, cytochrome c oxidase (COX). Immunohistochemistry for COX 1 protein demonstrated that, in response to cisplatin, expression was mostly maintained in focally damaged tubular epithelium. In contrast, COX enzyme activity in proximal tubules (by light microscopy) was decreased. Ultrastructural analysis of the cortex and outer stripe of the outer medulla showed decreased mitochondrial mass, disruption of cristae, and extensive mitochondrial swelling in proximal tubular epithelium. Functional electron microscopy showed that COX enzyme activity was decreased in the remaining mitochondria in the proximal tubules but maintained in distal tubules. In summary, cisplatin-induced nephrotoxicity is associated with structural and functional damage to the mitochondria. More broadly, using functional electron microscopy to measure mitochondrial enzyme activity may generate mechanistic insights across a spectrum of renal disorders.     

Neutralisation of Interleukin-13 in Mice Prevents Airway Pathology Caused by Chronic Exposure to House Dust Mite

Background

Repeated exposure to inhaled allergen can cause airway inflammation, remodeling and dysfunction that manifests as the symptoms of allergic asthma. We have investigated the role of the cytokine interleukin-13 (IL-13) in the generation and persistence of airway cellular inflammation, bronchial remodeling and deterioration in airway function in a model of allergic asthma caused by chronic exposure to the aeroallergen House Dust Mite (HDM).

Methodology/Principal Findings

Mice were exposed to HDM via the intranasal route for 4 consecutive days per week for up to 8 consecutive weeks. Mice were treated either prophylactically or therapeutically with a potent neutralising anti-IL-13 monoclonal antibody (mAb) administered subcutaneously (s.c.). Airway cellular inflammation was assessed by flow cytometry, peribronchial collagen deposition by histocytochemistry and airway hyperreactivity (AHR) by invasive measurement of lung resistance (R_L) and dynamic compliance (C_dyn). Both prophylactic and therapeutic treatment with an anti-IL-13 mAb significantly inhibited (P<0.05) the generation and maintenance of chronic HDM-induced airway cellular inflammation, peribronchial collagen deposition, epithelial goblet cell upregulation. AHR to inhaled methacholine was reversed by prophylactic but not therapeutic treatment with anti-IL-13 mAb. Both prophylactic and therapeutic treatment with anti-IL-13 mAb significantly reversed (P<0.05) the increase in baseline R_L and the decrease in baseline C_dyn caused by chronic exposure to inhaled HDM.

Conclusions/Significance

These data demonstrate that in a model of allergic lung disease driven by chronic exposure to a clinically relevant aeroallergen, IL-13 plays a significant role in the generation and persistence of airway inflammation, remodeling and dysfunction.     

The impact of the thermal sensitivity of cytochrome c oxidase on the respiration rate of Arctic charr red muscle mitochondria

To assess if cytochrome c oxidase could determine the response of mitochondrial respiration to changes in environmental temperature in ectotherms, we performed KCN titration of the respiration rate and cytochrome c oxidase activity in mitochondria from Arctic charr (Salvelinusfontinalis) muscle at four different temperatures (1 degrees C, 6 degrees C, 12 degrees C, and 18 degrees C). Our data showed an excess of cytochrome c oxidase activity over the mitochondrial state 3 respiration rate. Mitochondrial oxygen consumption rates reached approximately 12% of the cytochrome c oxidase maximal capacity at every temperature. Also, following titration, the mitochondrial respiration rate significantly decreased when KCN reached concentrations that inhibit almost 90% of the cytochrome c oxidase activity. This strongly supports the idea that the thermal sensitivity of the maximal mitochondrial respiration rate cannot be dictated by the effect of temperature on cytochrome c oxidase catalytic capacity. Furthermore, the strong similarity of the Q10s of mitochondrial respiration and cytochrome c oxidase activity suggests a functional or structural link between the two. The functional link could be coevolution of parts of the mitochondrial system to maintain optimal functions in most of the temperature range encountered by organisms.     

Cytochrome c oxidase activity in mitochondria of cardiomyocytes from isolated cardiac tissue incubated under long-term hypoxic conditions

Electron microscopic histochemistry based upon the oxidative polymerization of 3,3′-diaminobenzidine (DAB) was applied to identify cytochrome c oxidase activity. We found that the incubation of isolated small pieces of cardiac tissue over 72 h under hypoxic conditions caused changes in the mitochondrial ultrastructure and disorders in the functional activity of mitochondria, particularly in the IV complex of respirator chain. Small, electron-dense mitochondria appeared inside electron-light mitochondria (“mitochondria inside mitochondria”) stained positively for cytochrome c oxidase activity along the full length of crista. The results are discussed in connection with the concept of intracellular regeneration and mitochondria structural transformations during mitoptosis.     

Interaction of cytochrome c with mitochondrial proteins and cybacrone-dextran

Interaction of cytochrome c with electron carriers in intact and damaged (with destroyed outer membrane) rat liver mitochondria was studied. It was shown that the increase in ionic strength causes changes in the respiration rate of damaged mitochondria due to the reduction of the cytochrome c affinity for its binding sites in the organelles. This suggests that cytochrome c concentration in the intermembrane space of intact mitochondria is increased by salts, whereas the increase in ionic strength has a slight influence on the rates of succinate oxidase and external rotenone-insensitive NADH-oxidase of intact mitochondria. At low ionic strength values, the Michaelis constant (KM) value of external NADH-oxidase for cytochrome c exceeds by one order of magnitude that for succinate oxidase, while the maximal activity of these two systems is nearly the same. The increase in ionic strength causes an increase in the KM value for both oxidases. Interaction of cytochrome c with mitochondrial proteins was modelled by cytochrome c interaction with cibacron-dextran anions. It was concluded that the ionic strength-sensitive electrostatic interactions play a decisive role in cytochrome c binding to electron carriers in mitochondrial membranes. However, cytochrome c content and its binding parameters in intact-mitochondrial membranes prevent the latent activity of external NADH oxidase to be revealed in intact mitochondria after the increase in the ionic strength of the surrounding medium.     

The cytochrome c oxidase from Paracoccus denitrificans does not change the metal center ligation upon reduction

Cytochrome c oxidase catalyzes the reduction of oxygen to water. This process is accompanied by the vectorial transport of protons across the mitochondrial or bacterial membrane ("proton pumping"). The mechanism of proton pumping is still a matter of debate. Many proposed mechanisms require structural changes during the reaction cycle of cytochrome c oxidase. Therefore, the structure of the cytochrome c oxidase was determined in the completely oxidized and in the completely reduced states at a temperature of 100 K. No ligand exchanges or other major structural changes upon reduction of the cytochrome c oxidase from Paracoccus denitrificans were observed. The three histidine Cu(B) ligands are well defined in the oxidized and in the reduced states. These results are hardly compatible with the "histidine cycle" mechanisms formulated previously.     

The cytochrome c oxidase activity in mitochondria of cardiomyocytes of isolated cardiac tissue under long-term hypoxic incubation

Using method of electron microscopic histochemistry based upon the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to reveal cytochrome c oxidase activity we identified that long hypoxic incubation of isolated small pieces of cardiac tissue during 72 h caused changes in mitochondrial ultrastructure followed by a breach of functional activities of mitochondria, and, in particular, complex IV of the respiratory chain. But for all that, small electron-dense mitochondria appearing inside electron-light mitochondria ("mitochondria inside mitochondria") stained positively for cytochrome c oxidase activity along the full length of cristaes. The results obtained are discussed in connection with conception of changes in the mitochondrial reticulum ultrastructure during mitoptosis.     

Absorption spectra and magnetic circular dichroism of heme-containing proteins in nonequilibrium states. VI. Cytochrome c-oxidase

Low temperature (77 degrees K) absorption spectra of nonequilibrium states of cytochrome c oxidase produced by reduction of oxidases form protein by thermolysed electrons at 77 degrees K was studied. During reduction of cytochrome oxidase water-glycerol solution by thermolysed electrons at 77 degrees K a nonequilibrium reduced protein is formed. Low temperature (77 degrees K) absorption spectra of the nonequilibrium cytochrome oxidase differs from those reduced by ditionite. It was shown that the oxidation state of cytochrome a3 or addition of cytochrom c have no influence on these spectral changes. It is assumed, that the observed effects are conditioned by structural differences of reduced and oxidased cytochrome oxidase active center. Similar spectral changes were observed for cytochrome oxidase, bound to the mitochondrial membrane. At temperature increasing the low temperature reduced protein is relaxed to a corresponding equilibrium state. The spectral properties of bacterial cytochrome oxidase M. lysodeicticus do not depend on the way of reduction (by dytionite or thermolysed electrons at 77 degrees K).     

Mechanisms of cytochrome c release by proapoptotic BCL-2 family members

A crucial amplificatory event in several apoptotic cascades is the nearly complete release of cytochrome c from mitochondria. Proteins of the BCL-2 family which include both anti- and proapoptotic members control this step. Here, we review the proposed mechanisms by which proapoptotic BCL-2 family members induce cytochrome c release. Data support a model in which the apoptotic pathway bifurcates following activation of a "BH3 only" family member. BH3 only molecules induce the activation of the multidomain proapoptotics BAX and BAK, resulting in the permeabilization of the outer mitochondrial membrane and the efflux of cytochrome c. This is coordinated with the activation of a distinct pathway characterized by profound changes of the inner mitochondrial membrane morphology and organization. This mitochondrial remodelling insures complete release of cytochrome c and the onset of mitochondrial dysfunction that is a typical feature of many apoptotic deaths.     

The molecular mechanism of Noxa-induced mitochondrial dysfunction in p53-mediated cell death

Genotoxic stresses stabilize the p53 tumor suppressor protein which, in turn, transactivates target genes to cause apoptosis. Although Noxa, a "BH3-only" member of the Bcl-2 family, was shown to be a target of p53-mediated transactivation and to function as a mediator of p53-dependent apoptosis through mitochondrial dysfunction, the molecular mechanism by which Noxa causes mitochondrial dysfunction is largely unknown. Here we show that two domains (BH3 domain and mitochondrial targeting domain) in Noxa are essential for the release of cytochrome c from mitochondria. Noxa-induced cytochrome c release is inhibited by permeability transition pore inhibitors such as CsA or MgCl2, and Noxa induces an ultra-structural change of mitochondria yielding "swollen" mitochondria that are unlike changes induced by tBid. This indicates that Noxa may activate the permeability transition-related pore to release cytochrome c from mitochondria into cytosol. Moreover, Bak-oligomerization, which is an essential event for tBid-induced cytochrome c release in the extrinsic death signaling pathway, is not associated with Noxa-induced cytochrome c release. This finding suggests that the pathway of Noxa-induced mitochondrial dysfunction is distinct from the one of tBid-induced mitochondrial dysfunction. Thus, we propose that there are at least two different pathways of mitochondrial dysfunction; one mediated through Noxa in response to genotoxic stresses and the other through tBid in response to death ligands.     

Ischemia, rather than reperfusion, inhibits respiration through cytochrome oxidase in the isolated, perfused rabbit heart: role of cardiolipin

Ischemia and reperfusion result in mitochondrial dysfunction, with decreases in oxidative capacity, loss of cytochrome c, and generation of reactive oxygen species. During ischemia of the isolated perfused rabbit heart, subsarcolemmal mitochondria, located beneath the plasma membrane, sustain a loss of the phospholipid cardiolipin, with decreases in oxidative metabolism through cytochrome oxidase and the loss of cytochrome c. We asked whether additional injury to the distal electron chain involving cardiolipin with loss of cytochrome c and cytochrome oxidase occurs during reperfusion. Reperfusion did not lead to additional damage in the distal electron transport chain. Oxidation through cytochrome oxidase and the content of cytochrome c did not further decrease during reperfusion. Thus injury to cardiolipin, cytochrome c, and cytochrome oxidase occurs during ischemia rather than during reperfusion. The ischemic injury leads to persistent defects in oxidative function during the early reperfusion period. The decrease in cardiolipin content accompanied by persistent decrements in the content of cytochrome c and oxidation through cytochrome oxidase is a potential mechanism of additional myocyte injury during reperfusion.     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.     

Characterization and function of mitochondrial nitric-oxide synthase

The mitochondrial production of nitric oxide is catalyzed by a nitric-oxide synthase. This enzyme has the same cofactor and substrate requirements as other constitutive nitric-oxide synthases. Its occurrence was demonstrated in various mitochondrial preparations (intact, purified mitochondria, permeabilized mitochondria, mitoplasts, submitochondrial particles) from different organs (liver, heart) and species (rat, pig). Endogenous nitric oxide reversibly inhibits oxygen consumption and ATP synthesis by competitive inhibition of cytochrome oxidase. The increased K(m) of cytochrome oxidase for oxygen and the steady-state reduction of the electron chain carriers provided experimental evidence for the direct interaction of this oxidase with endogenous nitric oxide. The increase in hydrogen peroxide production by nitric oxide-producing mitochondria not accompanied by the full reduction of the respiratory chain components indicated that cytochrome c oxidase utilizes nitric oxide as an alternative substrate. Finally, effectors or modulators of cytochrome oxidase (the irreversible step in oxidative phosphorylation) had been proposed during the last 40 years. Nitric oxide is the first molecule that fulfills this role (it is a competitive inhibitor, produced at a fair rate near the target site) extending the oxygen gradient to tissues.     

Fractionation by differential and zonal centrifugation of spheroplasts prepared from a glucose-repressed fission yeast Schizosaccharomyces pombe 972h-.

A method is described for the preparation of spheroplasts in high yield from Schizosaccharomyces pombe, by treating cells grown in the presence of glucose and deoxyglucose with snail digestive enzymes. Gentle disruption of such spheroplasts yielded homogenates, from which marker enzymes for nuclei (NAD pyrophosphorylase) and mitochondria (cytochrome c oxidase activity and spectroscopically-detectable cytochromes a + a3) could be quantitatively sedimented by low-speed centrifugation. In contrast to previous findings with Saccharomyces carlsbergensis, cytochrome c oxidase and another mitochondrial enzyme, succinate dehydrogenase, were completely sedimentable by zonal centrifugation in sucrose gradients in the presence of either 2 mM-MgCl2 or 0-4 mM-EDTA. Mitochondria were apparently smaller and of lower buoyant density in gradients containing EDTA. The bulk of the total units of malate dehydrogenase and NADH; cytochrome c oxidoreductase sedimented with mitochondria, whereas NADPH: cytochrome c oxidoreductase was located in fractions containing no mitochondria. The distributions of mitochondrial enzymes were heterogeneous in populations of mitochondria separated on the basis of size or density. The possible origins of mitochondrial heterogeneity in extracts of S. pombe are discussed with special reference to changes in the enzyme activities of cells during the cell cycle.     

Decrease of cytochrome c oxidase protein in heart mitochondria of copper-deficient rats

Copper deficiency has been reported to be associated withdecreased cytochrome c oxidase activity, whichin turn may be responsible for theobserved mitochondrial impairment and cardiac failure. We isolatedmito-chondriafrom hearts of copper-deficient rats: cytochrome c oxidase activity was found to be lowerthan incopper-adequate mitochondria. The residual activity paralleled coppercontent of mitochondria and also corresponded with the heme amount associated with cytochromeaa3. In fact, lower absorption in thea-band region of cytochrome aa3 was foundfor copper-deficient rat heart mitochondria. Gel electrophoresisof protein extractedfrom mitochondrial membranes allowed measurements of protein content of thecomplexes ofoxidative phosphorylation, revealing a lower content of complex IV protein incopper-deficientrat heart mitochondria. The alterations caused by copper deficiency appear to bespecific forcytochrome c oxidase. Changes were not observed for F 0 F 1 ATP synthase activity,for heme contents ofcytochrome c and b, and for protein contents of complexes I, III and V.The present study demonstrates that the alteration of cytochrome c oxidase activityobserved in copper deficiency is due to a diminishedcontent of assembled protein and that shortnessof copper impairs heme insertion into cytochrome c oxidase.     

Cytochrome c oxidase is not a proton pump.

We conclude that the reduction of O2 to 2 H2O by cytochrome c oxidase of rat liver mitochondria involves the translocation of 4-from cytochrome c at the outer surface of the cristae membrane per O2 reduced and protonated by 4 H+ ions that enter the reaction domain from the inner aqueous phase. This net electron-translocating function of cytochrome c oxidase plugged through the mitochondrial cristae membrane is not linked to a proton-pumping function, such as that proposed by Wikstr?m [7,8].