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1
Fried, Nathan T., Cynthia Moffat, Erin L. Seifert, and Michael L. Oshinsky. "Functional mitochondrial analysis in acute brain sections from adult rats reveals mitochondrial dysfunction in a rat model of migraine." American Journal of Physiology-Cell Physiology 307, no. 11 (December 1, 2014): C1017—C1030. http://dx.doi.org/10.1152/ajpcell.00332.2013.
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Mitochondrial dysfunction has been implicated in many neurological disorders that only develop or are much more severe in adults, yet no methodology exists that allows for medium-throughput functional mitochondrial analysis of brain sections from adult animals. We developed a technique for quantifying mitochondrial respiration in acutely isolated adult rat brain sections with the Seahorse XF Analyzer. Evaluating a range of conditions made quantifying mitochondrial function from acutely derived adult brain sections from the cortex, cerebellum, and trigeminal nucleus caudalis possible. Optimization of this technique demonstrated that the ideal section size was 1 mm wide. We found that sectioning brains at physiological temperatures was necessary for consistent metabolic analysis of trigeminal nucleus caudalis sections. Oxygen consumption in these sections was highly coupled to ATP synthesis, had robust spare respiratory capacities, and had limited nonmitochondrial respiration, all indicative of healthy tissue. We demonstrate the effectiveness of this technique by identifying a decreased spare respiratory capacity in the trigeminal nucleus caudalis of a rat model of chronic migraine, a neurological disorder that has been associated with mitochondrial dysfunction. This technique allows for 24 acutely isolated sections from multiple brain regions of a single adult rat to be analyzed simultaneously with four sequential drug treatments, greatly advancing the ability to study mitochondrial physiology in adult neurological disorders.
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Gellerich, Frank Norbert, Sonata Trumbeckaite, Jens Rüdiger Opalka, Johannes Frank Gellerich, Ying Chen, Stephan Zierz, Karl Werdan, Christiane Neuhof, and Heinz Redl. "Mitochondrial Dysfunction in Sepsis: Evidence from Bacteraemic Baboons and Endotoxaemic Rabbits." Bioscience Reports 22, no. 1 (February 1, 2002): 99–113. http://dx.doi.org/10.1023/a:1016017224003.
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Mitochondria, that provide most of the ATP needed for cell work, and that play numerous specific functions in biosyntheses and degradations, as well as contributing to Ca2+; signaling, also play a key role in the pathway to cell death. Impairment of mitochondrial functions caused by mutations of mt-genome, and by acute processes, are responsible for numerous diseases. The involvement of impaired mitochondria in the pathogenesis of sepsis is discussed. By means of the skinned fiber technique and high resolution respirometry, we have detected significantly reduced rates of mitochondrial respiration in heart and skeletal muscle of endotoxaemic rabbits. Mitochondria from heart were more affected than those from skeletal muscle. Decreased respiration rates were accompanied by reduced activities of complex I+III of the respiratory chain. Endotoxin-caused impairment was also detectable at the level of the Langendorff perfused heart, where the coronary vascular resistance was significantly increased. For an investigation of the influence of bacteraemia on the mitochondrial respiratory chain, baboons were made septic by infusion of high and low amounts of E. coli. For complex I+III and II+III, a clear dose-dependent decrease was detectable and in animals which died in septic shock, a further decrease of enzyme activities in comparison to the controls were found. These results are discussed in the light of current knowledge on the role of mitochondria in cell pathology in respect to sepsis. In conclusion, we present evidence that mitochondrial function is disturbed during sepsis. Besides ischaemic and poison-induced disturbances of mitochondrial function, sepsis is a further example of an acute disease where impaired mitochondria have to be taken into account.
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Petrus, Alexandra, Corina Ratiu, Lavinia Noveanu, Rodica Lighezan, Mariana Rosca, Danina Muntean, and Oana Duicu. "Assessment of Mitochondrial Respiration in Human Platelets." Revista de Chimie 68, no. 4 (May 15, 2017): 768–71. http://dx.doi.org/10.37358/rc.17.4.5549.
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It has been long recognized that the impairment of platelet mitochondrial function occurs in a broad spectrum of diseases. Accordingly, the assessment of platelet respiratory dys/function has emerged as a putative approach allowing the characterization of the early impairment of human bioenergetic profile in several chronic pathologies. The aim of this study was to standardize the methodology for platelet isolation from peripheral blood and the measurement of mitochondrial oxygen consumption by means of high-resolution respirometry, respectively. The platelet isolation protocol consisted of two consecutive centrifugations of the whole blood collected from adult healthy females (n = 10) yielding a platelet-rich plasma sample. Respiration was measured at 370C using the Oxygraph-2k (Oroboros Instruments, Austria) according to a classic substrate-uncoupler-inhibitor-titration protocol. Platelets permeabilized with digitonin were allowed to respire in the presence of complex I (glutamate and malate) and complex II (succinate) substrates. We obtained a respiratory control ratio of 2.77 � 3.65 that indicates an accurate coupling efficiency of oxidative phosphorylation. The in vitro measurement of platelet respiration is a reliable method to evaluate the bioenergetic profile in humans. The standardized technique will be further used to assess the occurrence of mitochondrial dysfunction in peripheral blood in the setting of various chronic non-communicable diseases.
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Griffiths, E. J., and A. P. Halestrap. "Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion." Biochemical Journal 307, no. 1 (April 1, 1995): 93–98. http://dx.doi.org/10.1042/bj3070093.
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1. The yield of mitochondria isolated from perfused hearts subjected to 30 min ischaemia followed by 15 min reperfusion was significantly less than that for control hearts, and this was associated with a decrease in the rates of ADP-stimulated respiration. 2. The presence of 0.2 microM cyclosporin A (CsA) in the perfusion medium during ischaemia and reperfusion caused mitochondrial recovery to return to control values, but did not reverse the inhibition of respiration. 3. A technique has been devised to investigate whether the Ca(2+)-induced non-specific pore of the mitochondrial inner membrane opens during ischaemia and/or reperfusion of the isolated rat heart. The protocol involved loading the heart with 2-deoxy[3H]glucose ([3H]DOG), which will only enter mitochondria when the pore opens. Subsequent isolation of mitochondria demonstrated that [3H]DOG did not enter mitochondria during global isothermic ischaemia, but did enter during the reperfusion period. 4. The amount of [3H]DOG that entered mitochondria increased with the time of ischaemia, and reached a maximal value after 30-40 min of ischaemia. 5. CsA at 0.2 microM did not prevent [3H]DOG becoming associated with the mitochondria, but rather increased it; this was despite CsA having a protective effect on heart function similar to that shown previously [Griffiths and Halestrap (1993) J. Mol. Cell. Cardiol. 25, 1461-1469]. 6. The non-immunosuppressive CsA analogue [MeAla6]cyclosporin was shown to have a similar Ki to CsA on purified mitochondrial peptidyl-prolyl cis-trans-isomerase and mitochondrial pore opening, and also to have a similar protective effect against reperfusion injury. 7. Using isolated heart mitochondria, it was demonstrated that pore opening could become CsA-insensitive under conditions of adenine nucleotide depletion and high matrix [Ca2+] such as may occur during the initial phase of reperfusion. The apparent increase in mitochondrial [3H]DOG in the CsA-perfused hearts is explained by the ability of the drug to stabilize pore closure and so decrease the loss of [3H]DOG from the mitochondria during their preparation.
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Bednarczyk, Piotr, Agnieszka Koziel, Wieslawa Jarmuszkiewicz, and Adam Szewczyk. "Large-conductance Ca2+-activated potassium channel in mitochondria of endothelial EA.hy926 cells." American Journal of Physiology-Heart and Circulatory Physiology 304, no. 11 (June 1, 2013): H1415—H1427. http://dx.doi.org/10.1152/ajpheart.00976.2012.
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In the present study, we describe the existence of a large-conductance Ca2+-activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassium-selective current was recorded with a mean conductance equal to 270 ± 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 μM Ca2+, 10 μM NS1619, and 0.5 μM NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassium-dependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a pore-forming α-subunit and an auxiliary β2-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calcium-dependent potassium channel with properties similar to those of the surface membrane BKCa channel.
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Naveenan, Manoharan, Rolf Erik Olsen, and Bjørg Egelandsdal. "Effects of dietary glutamate and succinate on growth performance and mitochondrial respiration in heart and liver of Atlantic salmon (Salmo salar) smolts." Journal of Fisheries 7, no. 2 (June 3, 2019): 692–99. http://dx.doi.org/10.17017/j.fish.2.
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The smolt stage of salmon has challenges in reaching adequate growth rates due to the changing environmental conditions at sea. Therefore, it is necessary to provide adequate diets to achieve sufficient growth. This study determined the impacts of glutamate and succinate (1% each) supplemented diet on the growth of Atlantic salmon smolts along with characterization of mitochondrial respiration using high-resolution respirometry technique. Results indicated that there was no significant difference in growth response between the treatment and control groups. Maximum oxidative phosphorylation (OXPHOS) was reached after addition of succinate. Analysis of heart homogenates revealed a significant difference in LEAK respiration state (P = 0.005). No significant difference was recorded between the diet groups for liver homogenates. Differences between heart and liver respiration revealed that mitochondrial activity is organ dependent.
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Guerrero, Karen, Bernard Wuyam, Paulette Mezin, Isabelle Vivodtzev, Marko Vendelin, Jean-Christian Borel, Rachid Hacini, et al. "Functional coupling of adenine nucleotide translocase and mitochondrial creatine kinase is enhanced after exercise training in lung transplant skeletal muscle." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 289, no. 4 (October 2005): R1144—R1154. http://dx.doi.org/10.1152/ajpregu.00229.2005.
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Mechanisms responsible for limitation of exercise capacity in lung transplant recipients (LR) and benefits gained by exercise training were studied. Mitochondrial respiration parameters, energy transfer, and cell structure were assessed in vastus lateralis biopsies using the permeabilized fiber technique with histochemical and morphometric measurements. Twelve male controls (C) and 12 LR performed exercise training over 12 wk. Before exercise training, there were strong correlations between exercise capacity (maximal O2 consumption and endurance time at 70% maximal power output) and cellular events, as assessed by percentage of type I fibers and apparent Km for exogenous ADP. Anticalcineurins were not involved in LR exercise limitation, since there were no differences in maximal mitochondrial rate of respiration before exercise training and no abnormalities in respiratory chain complexes compared with C. Training resulted in a significant increase in physiological parameters both at the cellular (apparent Km for exogenous ADP and stimulating effect of creatine) and integrated (maximal O2 consumption, power output at ventilatory threshold, maximal power output, and endurance time at 70% maximal power output) levels in LR and C. After the training period, improvements in maximal O2 consumption and in maximal mitochondrial rate of respiration were noted, as well as changes in endurance time and percentage of type I fibers. Because there were no changes in diameters and fiber types, baseline alteration of apparent Km for exogenous ADP and its improvement after training might be related to changes within the intracellular energetic units. After the training period, intracellular energetic units exhibited a higher control of mitochondrial respiration by creatine linked to a more efficient functional coupling adenine nucleotide translocase-mitochondrial creatine kinase, resulting in better exercise performances in C and LR.
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Weinberg, Guy L., June W. Palmer, Timothy R. VadeBoncouer, Mikko B. Zuechner, Guy Edelman, and Charles L. Hoppel. "Bupivacaine Inhibits Acylcarnitine Exchange in Cardiac Mitochondria." Anesthesiology 92, no. 2 (February 1, 2000): 523. http://dx.doi.org/10.1097/00000542-200002000-00036.
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Background The authors previously reported that secondary carnitine deficiency may sensitize the heart to bupivacaine-induced arrhythmias. In this study, the authors tested whether bupivacaine inhibits carnitine metabolism in cardiac mitochondria. Methods Rat cardiac interfibrillar mitochondria were prepared using a differential centrifugation technique. Rates of adenosine diphosphate-stimulated (state III) and adenosine diphosphate-limited (state IV) oxygen consumption were measured using a Clark electrode, using lipid or nonlipid substrates with varying concentrations of a local anesthetic. Results State III respiration supported by the nonlipid substrate pyruvate (plus malate) is minimally affected by bupivacaine concentrations up to 2 mM. Lower concentrations of bupivacaine inhibited respiration when the available substrates were palmitoylcarnitine or acetylcarnitine; bupivacaine concentration causing 50% reduction in respiration (IC50 +/- SD) was 0.78+/-0.17 mM and 0.37+/-0.03 mM for palmitoylcarnitine and acetylcarnitine, respectively. Respiration was equally inhibited by bupivacaine when the substrates were palmitoylcarnitine alone, or palmitoyl-CoA plus carnitine. Bupivacaine (IC50 = 0.26+/-0.06 mM) and etidocaine (IC50 = 0.30+/-0.12 mM) inhibit carnitine-stimulated pyruvate oxidation similarly, whereas the lidocaine IC50 is greater by a factor of roughly 5, (IC50 = 1.4+/-0.26 mM), and ropivacaine is intermediate, IC50 = 0.5+/-0.28 mM. Conclusions Bupivacaine inhibits mitochondrial state III respiration when acylcarnitines are the available substrate. The substrate specificity of this effect rules out bupivacaine inhibition of carnitine palmitoyl transferases I and II, carnitine acetyltransferase, and fatty acid beta-oxidation. The authors hypothesize that differential inhibition of carnitine-stimulated pyruvate oxidation by various local anesthetics supports the clinical relevance of inhibition of carnitine-acylcarnitine translocase by local anesthetics with a cardiotoxic profile.
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McCrimmon, Allison, Mark Domondon, Regina F. Sultanova, Daria V. Ilatovskaya, and Krisztian Stadler. "Comprehensive assessment of mitochondrial respiratory function in freshly isolated nephron segments." American Journal of Physiology-Renal Physiology 318, no. 5 (May 1, 2020): F1237—F1245. http://dx.doi.org/10.1152/ajprenal.00031.2020.
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Changes in mitochondrial function are central to many forms of kidney disease, including acute injury, diabetic nephropathy, hypertension, and chronic kidney diseases. As such, there is an increasing need for reliable and fast methods for assessing mitochondrial respiratory function in renal cells. Despite being indispensable for many mechanistic studies, cultured cells or isolated mitochondria, however, often do not recapitulate in vivo or close to in vivo situations. Cultured and/or immortalized cells often change their bioenergetic profile and phenotype compared with in vivo or ex vivo situations, and isolated mitochondria are simply removed from their cellular milieu. This is especially important for extremely complex organs such as the kidney. Here, we report the development and validation of a new approach for the rapid assessment of mitochondrial oxygen consumption on freshly isolated glomeruli or proximal tubular fragments using Agilent SeaHorse XFe24 and XF96 Extracellular Flux Analyzers. We validated the technique in several healthy and diseased rodent models: the C57BL/6J mouse, the diabetic db/ db mouse and matching db/+ control mouse, and the Dahl salt-sensitive rat. We compared the data to respiration from isolated mitochondria. The method can be adapted and used for the rapid assessment of mitochondrial oxygen consumption from any rodent model of the investigator’s choice. The isolation methods presented here ensure viable and functional proximal tubular fragments and glomeruli, with a preserved cellular environment for studying mitochondrial function within the context of their surroundings and interactions.
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Zhdanov, Alexander V., Ruslan I. Dmitriev, and Dmitri B. Papkovsky. "Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling." Bioscience Reports 32, no. 6 (October 5, 2012): 587–95. http://dx.doi.org/10.1042/bsr20120085.
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Mitochondrial uncoupling is implicated in many patho(physiological) states. Using confocal live cell imaging and an optical O2 sensing technique, we show that moderate uncoupling of the mitochondria with plecomacrolide Baf (bafilomycin A1) causes partial depolarization of the mitochondria and deep sustained deoxygenation of human colon cancer HCT116 cells subjected to 6% atmospheric O2. A decrease in iO2 (intracellular O2) to 0–10 μM, induced by Baf, is sufficient for stabilization of HIFs (hypoxia inducible factors) HIF-1α and HIF-2α, coupled with an increased expression of target genes including GLUT1 (glucose transporter 1), HIF PHD2 (prolyl hydroxylase domain 2) and CAIX (carbonic anhydrase IX). Under the same hypoxic conditions, treatment with Baf causes neither decrease in iO2 nor HIF-α stabilization in the low-respiring HCT116 cells deficient in COX (cytochrome c-oxidase). Both cell types display equal capacities for HIF-α stabilization by hypoxia mimetics DMOG (dimethyloxalylglycine) and CoCl2, thus suggesting that the effect of Baf under hypoxia is driven mainly by mitochondrial respiration. Altogether, by activating HIF signalling under moderate hypoxia, mitochondrial uncoupling can play an important regulatory role in colon cancer metabolism and modulate adaptation of cancer cells to natural hypoxic environments.
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Nowak, Grazyna, Diana Bakajsova, and Allen M. Samarel. "Protein kinase C-ε activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules." American Journal of Physiology-Renal Physiology 301, no. 1 (July 2011): F197—F208. http://dx.doi.org/10.1152/ajprenal.00364.2010.
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PKC-ε activation mediates protection from ischemia-reperfusion injury in the myocardium. Mitochondria are a subcellular target of these protective mechanisms of PKC-ε. Previously, we have shown that PKC-ε activation is involved in mitochondrial dysfunction in oxidant-injured renal proximal tubular cells (RPTC; Nowak G, Bakajsova D, Clifton GL Am J Physiol Renal Physiol 286: F307–F316, 2004). The goal of this study was to examine the role of PKC-ε activation in mitochondrial dysfunction and to identify mitochondrial targets of PKC-ε in RPTC. The constitutively active and inactive mutants of PKC-ε were overexpressed in primary cultures of RPTC using the adenoviral technique. Increases in active PKC-ε levels were accompanied by PKC-ε translocation to mitochondria. Sustained PKC-ε activation resulted in decreases in state 3 respiration, electron transport rate, ATP production, ATP content, and activities of complexes I and IV and F0F1-ATPase. Furthermore, PKC-ε activation increased mitochondrial membrane potential and oxidant production and induced mitochondrial fragmentation and RPTC death. Accumulation of the dynamin-related protein in mitochondria preceded mitochondrial fragmentation. Antioxidants blocked PKC-ε-induced increases in the oxidant production but did not prevent mitochondrial fragmentation and cell death. The inactive PKC-ε mutant had no effect on mitochondrial functions, morphology, oxidant production, and RPTC viability. We conclude that active PKC-ε targets complexes I and IV and F0F1-ATPase in RPTC. PKC-ε activation mediates mitochondrial dysfunction, hyperpolarization, and fragmentation. It also induces oxidant generation and cell death, but oxidative stress is not the mechanism of RPTC death. These results show that in contrast to protective effects of PKC-ε activation in cardiomyocytes, sustained PKC-ε activation is detrimental to mitochondrial function and viability in RPTC.
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Kuznetsov, Andrey V., Rita Guzun, François Boucher, Rafaela Bagur, Tuuli Kaambre, and Valdur Saks. "Mysterious Ca2+-independent muscular contraction: déjà vu." Biochemical Journal 445, no. 3 (July 13, 2012): 333–36. http://dx.doi.org/10.1042/bj20120439.
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The permeabilized cells and muscle fibres technique allows one to study the functional properties of mitochondria without their isolation, thus preserving all of the contacts with cellular structures, mostly the cytoskeleton, to study the whole mitochondrial population in the cell in their natural surroundings and it is increasingly being used in both experimental and clinical studies. The functional parameters (affinity for ADP in regulation of respiration) of mitochondria in permeabilized myocytes or myocardial fibres are very different from those in isolated mitochondria in vitro. In the present study, we have analysed the data showing the dependence of this parameter upon the muscle contractile state. Most remarkable is the effect of recently described Ca2+-independent contraction of permeabilized muscle fibres induced by elevated temperatures (30–37°C). We show that very similar strong spontaneous Ca2+-independent contraction can be produced by proteolytic treatment of permeabilized muscle fibres that result in a disorganization of mitochondrial arrangement, leading to a significant increase in affinity for ADP. These data show that Ca2+-insensitive contraction may be related to the destruction of cytoskeleton structures by intracellular proteases. Therefore the use of their inhibitors is strongly advised at the permeabilization step with careful washing of fibres or cells afterwards. A possible physiologically relevant relationship between Ca2+-regulated ATP-dependent contraction and mitochondrial functional parameters is also discussed.
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Supinski, G. S., M. P. Murphy, and L. A. Callahan. "MitoQ administration prevents endotoxin-induced cardiac dysfunction." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 297, no. 4 (October 2009): R1095—R1102. http://dx.doi.org/10.1152/ajpregu.90902.2008.
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Sepsis elicits severe alterations in cardiac function, impairing cardiac mitochondrial and pressure-generating capacity. Currently, there are no therapies to prevent sepsis-induced cardiac dysfunction. We tested the hypothesis that administration of a mitochondrially targeted antioxidant, 10-(6′-ubiquinonyl)-decyltriphenylphosphonium (MitoQ), would prevent endotoxin-induced reductions in cardiac mitochondrial and contractile function. Studies were performed on adult rodents ( n = 52) given either saline, endotoxin (8 mg·kg−1·day−1), saline + MitoQ (500 μM), or both endotoxin and MitoQ. At 48 h animals were killed and hearts were removed for determination of either cardiac mitochondrial function (using polarography) or cardiac pressure generation (using the Langendorf technique). We found that endotoxin induced reductions in mitochondrial state 3 respiration rates, the respiratory control ratio, and ATP generation. Moreover, MitoQ administration prevented each of these endotoxin-induced abnormalities, P < 0.001. We also found that endotoxin produced reductions in cardiac pressure-generating capacity, reducing the systolic pressure-diastolic relationship. MitoQ also prevented endotoxin-induced reductions in cardiac pressure generation, P < 0.01. One potential link between mitochondrial and contractile dysfunction is caspase activation; we found that endotoxin increased cardiac levels of active caspases 9 and 3 ( P < 0.001), while MitoQ prevented this increase ( P < 0.01). These data demonstrate that MitoQ is a potent inhibitor of endotoxin-induced mitochondrial and cardiac abnormalities. We speculate that this agent may prove a novel therapy for sepsis-induced cardiac dysfunction.
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Irina, Zhigacheva, Volodkin Aleksandr, and Rasulov Maksud. "The Influence of Ultra-Low Concentrations of Potassium Anphen on the Bioenergetic Characteristics of Mitochondria." Current Bioactive Compounds 16, no. 4 (June 19, 2020): 537–42. http://dx.doi.org/10.2174/1573407214666181116093909.
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Background: One of the main sources of ROS in stress conditions is the mitochondria. Excessive generation of ROS leads to oxidation of thiol groups of proteins, peroxidation of membrane lipids and swelling of the mitochondria. In this regard, there is a need to search for preparationsadaptogens that increase the body's resistance to stress factors. Perhaps, antioxidants can serve as such adaptogens. This work aims at studying the effect of antioxidant; the potassium anphen in a wide range of concentrations on the functional state of 6 day etiolated pea seedlings mitochondria (Pisum sativum L). Methods: The functional state of mitochondria was studied per rates of mitochondria respiration, by the level of lipid peroxidation and study of fatty acid composition of mitochondrial membranes by chromatography technique. Results: Potassium anphen in concentrations of 10-5 - 10-8 M and 10-13-10-16 prevented the activation of LPO in the mitochondrial membranes of pea seedlings, increased the oxidation rates of NAD-dependent substrates and succinate in the respiratory chain of mitochondria that probably pointed to the anti-stress properties of the drug. Indeed, the treatment of pea seeds with the preparation in concentrations of 10-13 M prevented the inhibition of growth of seedlings in conditions of water deficiency. Conclusion: It is assumed that the dose dependence of the biological effects of potassium anphen and the manifestation of these effects in ultra-low concentrations are due to its ability in water solutions to form a hydrate containing molecular ensembles (structures).
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Mallatt, Jon, Steven J. Lampa, J. Franklin Bailey, Marc A. Evans, and Steve Brumbaugh. "A fish gill system for quantifying the ultrastructural effects of environmental stressors: methylmercury, Kepone®, and heat shock." Canadian Journal of Fisheries and Aquatic Sciences 52, no. 6 (June 1, 1995): 1165–82. http://dx.doi.org/10.1139/f95-113.
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In previous histopathological studies, fish gills have been used to study nonspecific responses of tissue to injury; however, such studies have revealed little about the specific effects of different toxicants on cells. Here, we used transmission electron microscopy plus a morphometric technique (stereology) to determine how several types of gill cells are specifically altered by three stressors: methylmercury, heat shock, and the insecticide Kepone® (chlordecone). Larval sea lampreys, Petromyzon marinus, were exposed to various concentrations of waterborne stressor (ranging from the incipient lethal concentrations to a 24-h LC50) for different periods (half a day or 3 weeks). Methylmercury increased the volumes of gill epithelial cells and their nuclei and mitochondria, presumably by inducing ambient water to leak into these structures. Heat shock, by contrast, decreased the volumes of most cells and their organelles, presumably by causing cells to shed pieces of cytoplasm. Kepone had almost no effect, but the few changes that were recorded (increased mitochondrial volume in one cell type) are consistent with known actions of Kepone® (alteration of mitochondrial respiration). The results prove that a fish gill can be used to probe the specific actions of different toxicants and pollutants.
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Chen, X. J., and G. D. Clark-Walker. "Mutations in MGI genes convert Kluyveromyces lactis into a petite-positive yeast." Genetics 133, no. 3 (March 1, 1993): 517–25. http://dx.doi.org/10.1093/genetics/133.3.517.
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Abstract Following targeted disruption of the unique CYC1 gene, the petite-negative yeast, Kluyveromyces lactis, was found to grow fermentatively in the absence of cytochrome c-mediated respiration. This observation encouraged us to seek mitochondrial mutants by treatment of K. lactis with ethidium bromide at the highest concentration permitting survival. By this technique, we isolated four mtDNA mutants, three lacking mtDNA and one with a deleted mitochondrial genome. In the three isolates lacking mtDNA, a nuclear mutation is present that permits petite formation. The three mutations occur at two different loci, designated MGI1 and MGI2 (for Mitochondrial Genome Integrity). The mgi mutations convert K. lactis into a petite-positive yeast. Like bakers' yeast, the mgi mutants spontaneously produce petites with deletions in mtDNA and lose this genome at high frequency on treatment with ethidium bromide. We suggest that the MGI gene products are required for maintaining the integrity of the mitochondrial genome and that, petite-positive yeasts may be naturally altered in one or other of these genes.
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Caldeira, Diego Elias da Silva, Maria Eliza Jordani Souza, Maria Cecília Jordani Gomes, Maria Aparecida Neves Cardoso Picinato, Clarice Fleury Fina, Omar Feres, and Orlando Castro e Silva. "Effects of hyperbaric oxygen (HBO), as pre-conditioning in liver of rats submitted to periodic liver ischemia/reperfusion." Acta Cirurgica Brasileira 28, suppl 1 (2013): 66–71. http://dx.doi.org/10.1590/s0102-86502013001300013.
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PURPOSE: to assess the effect of hyperbaric oxygen (HBO) as pre-conditioning on periodic liver ischemia/reperfusion injury. METHODS: Thirty-six male Wistar rats were divided into 4 groups (SHAM, I/R , HBO-I/R and CONTROL). The surgical technique consisted of total clamping of the hepatic pedicle for 15 min followed by twice repeated reperfusion for 5 min (unclamping). HBO was applied in a collective chamber (simultaneous exposure of 4 rats) directly pressurized with oxygen at 2 ATA for 60 min. Hepatic mitochondrial function was determined using samples of the median lobe obtained after exactly 5 min of reperfusion for the analysis of mitochondrial respiration based on the determination of states 3 and 4, the respiratory control ratio and the transition of mitochondrial permeability (mitochondrial swelling).Data were analyzed by the Mann-Whitney test and the level of significance was set at p < 0.05. RESULTS: There was a statistically significant difference (p< 0.05) in state 3 between the CONTROL and I/R and HBO-I/R groups, in state 4 between the CONTROL and I/R and HBO-I/R groups; in respiratory control ratio (RCR) between the CONTROL and I/R and HBO-I/R groups and between the CONTROL and Sham groups, and in mitochondrial swelling between the CONTROL and I/R and HBO-/R groups and between the Sham and I/R and HBO-I/R groups. CONCLUSION: In this process of periodic ischemia and reperfusion, hyperbaric pre-conditioning did not improve significantly hepatic mitochondrial function.
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Kaczara, Patrycja, Barbara Sitek, Kamil Przyborowski, Anna Kurpinska, Kamil Kus, Marta Stojak, and Stefan Chlopicki. "Antiplatelet Effect of Carbon Monoxide Is Mediated by NAD + and ATP Depletion." Arteriosclerosis, Thrombosis, and Vascular Biology 40, no. 10 (October 2020): 2376–90. http://dx.doi.org/10.1161/atvbaha.120.314284.
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Objectives: Carbon monoxide (CO) produced by haem oxygenases or released by CO-releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO–induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry–based metabolomics were used. CORM-A1–induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1–induced delay of tricarboxylic acid cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD + ) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH [glyceraldehyde 3-phosphate dehydrogenase]), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD + –dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1–induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD + regeneration, pointing out to the key role of NAD + depletion in the inhibition of platelet bioenergetics by CORM-A1. Conclusions: The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration—attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis—ascribed to cytosolic NAD + depletion.
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Ohnishi, Tomoko, S. Tsuyoshi Ohnishi, and John C. Salerno. "Five decades of research on mitochondrial NADH-quinone oxidoreductase (complex I)." Biological Chemistry 399, no. 11 (October 25, 2018): 1249–64. http://dx.doi.org/10.1515/hsz-2018-0164.
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AbstractNADH-quinone oxidoreductase (complex I) is the largest and most complicated enzyme complex of the mitochondrial respiratory chain. It is the entry site into the respiratory chain for most of the reducing equivalents generated during metabolism, coupling electron transfer from NADH to quinone to proton translocation, which in turn drives ATP synthesis. Dysfunction of complex I is associated with neurodegenerative diseases such as Parkinson’s and Alzheimer’s, and it is proposed to be involved in aging. Complex I has one non-covalently bound FMN, eight to 10 iron-sulfur clusters, and protein-associated quinone molecules as electron transport components. Electron paramagnetic resonance (EPR) has previously been the most informative technique, especially in membranein situanalysis. The structure of complex 1 has now been resolved from a number of species, but the mechanisms by which electron transfer is coupled to transmembrane proton pumping remains unresolved. Ubiquinone-10, the terminal electron acceptor of complex I, is detectable by EPR in its one electron reduced, semiquinone (SQ) state. In the aerobic steady state of respiration the semi-ubiquinone anion has been observed and studied in detail. Two distinct protein-associated fast and slow relaxing, SQ signals have been resolved which were designated SQNfand SQNs. This review covers a five decade personal journey through the field leading to a focus on the unresolved questions of the role of the SQ radicals and their possible part in proton pumping.
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Takahashi, Eiji, and Michihiko Sato. "Imaging of oxygen gradients in monolayer cultured cells using green fluorescent protein." American Journal of Physiology-Cell Physiology 299, no. 6 (December 2010): C1318—C1323. http://dx.doi.org/10.1152/ajpcell.00254.2010.
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Gradients of Po2 between capillary blood and mitochondria are the driving force for diffusional O2 delivery in tissues. Hypoxic microenvironments in tissues that result from diffusional O2 gradients are especially relevant in solid tumors because they have been related to a poor prognosis. To address the impact of tissue O2 gradients, we developed a novel technique that permits imaging of intracellular O2 levels in cultured cells at a subcellular spatial resolution. This was done, with the sensitivity to O2 ≤3%, by the O2-dependent red shift of green fluorescent protein (AcGFP1) fluorescence. Measurements were carried out in a confluent monolayer of Hep3B cells expressing AcGFP1 in the cytoplasm. To establish a two-dimensional O2 diffusion model, a thin quartz glass slip was placed onto the monolayer cells to prevent O2 diffusion from the top surface of the cell layer. The magnitude of the red shift progressively increased as the distance from the gas coverslip interface increased. It reached an anoxic level in cells located at ∼220 μm and ∼690 μm from the gas coverslip boundary at 1% and 3% gas phase O2, respectively. Thus the average O2 gradient was 0.03 mmHg/μm in the present tissue model. Abolition of mitochondrial respiration significantly dampened the gradients. Furthermore, intracellular gradients of the red shift in mitochondria-targeted AcGFP1 in single Hep3B cells suggest that the origin of tissue O2 gradients is intracellular. Findings in the present two-dimensional O2 diffusion model support the crucial role of tissue O2 diffusion in defining the O2 microenvironment in individual cells.
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Fongy, Anaïs, Caroline Romestaing, Coralie Blanc, Nicolas Lacoste-Garanger, Jean-Louis Rouanet, Mireille Raccurt, and Claude Duchamp. "Ontogeny of muscle bioenergetics in Adélie penguin chicks (Pygoscelis adeliae)." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 305, no. 9 (November 1, 2013): R1065—R1075. http://dx.doi.org/10.1152/ajpregu.00137.2013.
Full textAbstract:
The ontogeny of pectoralis muscle bioenergetics was studied in growing Adélie penguin chicks during the first month after hatching and compared with adults using permeabilized fibers and isolated mitochondria. With pyruvate-malate-succinate or palmitoyl-carnitine as substrates, permeabilized fiber respiration markedly increased during chick growth (3-fold) and further rose in adults (1.4-fold). Several markers of muscle fiber oxidative activity (cytochrome oxidase, citrate synthase, hydroxyl-acyl-CoA dehydrogenase) increased 6- to 19-fold with age together with large rises in intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial content (3- to 5-fold) and oxidative activities (1.5- to 2.4-fold). The proportion of IMF relative to SS mitochondria increased with chick age but markedly dropped in adults. Differences in oxidative activity between mitochondrial fractions were reduced in adults compared with hatched chicks. Extrapolation of mitochondrial to muscle respirations revealed similar figures with isolated mitochondria and permeabilized fibers with carbohydrate-derived but not with lipid-derived substrates, suggesting diffusion limitations of lipid substrates with permeabilized fibers. Two immunoreactive fusion proteins, mitofusin 2 (Mfn2) and optic atrophy 1 (OPA1), were detected by Western blots on mitochondrial extracts and their relative abundance increased with age. Muscle fiber respiration was positively related with Mfn2 and OPA1 relative abundance. Present data showed by two complementary techniques large ontogenic increases in muscle oxidative activity that may enable birds to face thermal emancipation and growth in childhood and marine life in adulthood. The concomitant rise in mitochondrial fusion protein abundance suggests a role of mitochondrial networks in the skeletal muscle processes of bioenergetics that enable penguins to overcome harsh environmental constraints.
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Serteyn, D., J. Ceusters, S. Nonnenmacher, K. Kirsch, A. Mouithys-Mickalad, T. Franck, J. P. Lejeune, and C. Sandersen. "Mitochondrial function and aerobic capacity assessed by high resolution respirometry in Thoroughbred horses." Comparative Exercise Physiology 12, no. 2 (June 24, 2016): 67–73. http://dx.doi.org/10.3920/cep150031.
Full textAbstract:
During the initial stages of training of young Thoroughbred horses, low intensity exercise is employed to increase aerobic capacity. High Resolution Respirometry (HRR) allows the determination of aerobic capacities in small samples of permeabilised muscle fibres. The aim of the study was to measure the mitochondrial function by HRR in Thoroughbred horses, to compare these values to Warmblood horses and to evaluate the effect of a 10-weeks training period. The mitochondrial function was measured by HRR using different substrate-uncoupler protocols (SUIT 1 and 2) in muscle microbiopsies from two groups of untrained horses: 17 Warmblood and 8 Thoroughbred and in the group of 8 Thoroughbred horses before and after a 10-week training period. The SUIT1 protocol employed to compare the two groups of horses showed that in Thoroughbred horses, the mean values for oxygen flux expressed as tissue mass-specific respiration were significantly higher for complex I (CI)Glutamate+Malate, CI + complex II, and maximum electron transport capacities (ETSmax) than the mean values measured in Warmblood horses. The SUIT 1 and SUIT 2 protocols revealed large differences among Thoroughbred horses before and after training. The SUIT 2 protocols showed a significant difference for the complex I activity before and after training but only when the oxygen flux was expressed as percentage of ETSmax. This study shows the interest of HRR in equine sport medicine and exercise physiology, but shows that the technique requires further refinement. Indeed significant differences have been shown between the Thoroughbred and the Warmblood horses highlighting the need to have baseline data for each breed. The Thoroughbred horses had globally a high oxidative phosphorylation capacity with an increase of CI activity induced by an aerobic training program.
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Marchini, Timoteo, Verónica D'Annunzio, Mariela L. Paz, Lourdes Cáceres, Mariana Garcés, Virginia Perez, Deborah Tasat, et al. "Selective TNF-α targeting with infliximab attenuates impaired oxygen metabolism and contractile function induced by an acute exposure to air particulate matter." American Journal of Physiology-Heart and Circulatory Physiology 309, no. 10 (November 15, 2015): H1621—H1628. http://dx.doi.org/10.1152/ajpheart.00359.2015.
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Inflammation plays a central role in the onset and progression of cardiovascular diseases associated with the exposure to air pollution particulate matter (PM). The aim of this work was to analyze the cardioprotective effect of selective TNF-α targeting with a blocking anti-TNF-α antibody (infliximab) in an in vivo mice model of acute exposure to residual oil fly ash (ROFA). Female Swiss mice received an intraperitoneal injection of infliximab (10 mg/kg body wt) or saline solution, and were intranasally instilled with a ROFA suspension (1 mg/kg body wt). Control animals were instilled with saline solution and handled in parallel. After 3 h, heart O2 consumption was assessed by high-resolution respirometry in left ventricle tissue cubes and isolated mitochondria, and ventricular contractile reserve and lusitropic reserve were evaluated according to the Langendorff technique. ROFA instillation induced a significant decrease in tissue O2 consumption and active mitochondrial respiration by 32 and 31%, respectively, compared with the control group. While ventricular contractile state and isovolumic relaxation were not altered in ROFA-exposed mice, impaired contractile reserve and lusitropic reserve were observed in this group. Infliximab pretreatment significantly attenuated the decrease in heart O2 consumption and prevented the decrease in ventricular contractile and lusitropic reserve in ROFA-exposed mice. Moreover, infliximab-pretreated ROFA-exposed mice showed conserved left ventricular developed pressure and cardiac O2 consumption in response to a β-adrenergic stimulus with isoproterenol. These results provides direct evidence linking systemic inflammation and altered cardiac function following an acute exposure to PM and contribute to the understanding of PM-associated cardiovascular morbidity and mortality.
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Raddatz, E., U. Katz, and P. Kucera. "Oxygen uptake of isolated toad skin epithelium: micromeasurement and effect of ionic acclimation." American Journal of Physiology-Cell Physiology 260, no. 5 (May 1, 1991): C1117—C1124. http://dx.doi.org/10.1152/ajpcell.1991.260.5.c1117.
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Oxidative metabolism of isolated toad skin epithelium (Bufo viridis) was investigated in vitro under open-circuit conditions using the spectrophotometric oxyhemoglobin micromethod. This highly sensitive technique has been adapted for studying several epithelia in parallel and for detecting possible regional variations of oxygen uptake in individual epithelium. Changes in the proportion of mitochondria-rich cells (MRC) by ionic acclimation affected oxidative metabolism under nontransporting condition. After acclimation of animals to either NaNO3 or NaCl solutions (100 mmol/l, for greater than 2 wk), the number of MRC per square millimeter in epithelia from nonacclimated and NaNO3- and NaCl-acclimated animals was 350 +/- 113, 460 +/- 196, and 107 +/- 52, respectively. O2 uptake of nonacclimated and NaNO3-acclimated epithelia was significantly higher than that of NaCl-acclimated epithelia (i.e., 0.89 and 0.90 vs. 0.57 nmol O2.h-1.mm-2, respectively). The correlation established between O2 uptake and number of MRC allowed evaluation of the respiration rate of one single MRC, i.e., approximately 1 pmol O2/h. The lowest mitochondrial oxidative activity was found in the epithelia from NaCl-acclimated toads where the uncoupler 2,4-dinitrophenol (50 mumols/l) had the highest relative stimulatory effect (+114%). Acetazolamide (50 mumols/l), a potent inhibitor of carbonic anhydrase mainly present in the MRC, reduced selectively by 31% O2 uptake of the MRC-rich epithelia (NaNO3 acclimated). O2 uptake increased significantly by approximately 80% when basolateral pH increased from 5.8 to 7.8, but did not depend on apical pH. These findings indicate that under nontransporting (open-circuit) conditions, aerobic metabolism of the isolated toad skin epithelium is related to the density and/or characteristics of the MRC.(ABSTRACT TRUNCATED AT 250 WORDS)
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Walewska, Agnieszka, Milena Krajewska, Aleksandra Stefanowska, Aleksandra Buta, Renata Bilewicz, Paweł Krysiński, Piotr Bednarczyk, Piotr Koprowski, and Adam Szewczyk. "Methods of Measuring Mitochondrial Potassium Channels: A Critical Assessment." International Journal of Molecular Sciences 23, no. 3 (January 21, 2022): 1210. http://dx.doi.org/10.3390/ijms23031210.
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In this paper, the techniques used to study the function of mitochondrial potassium channels are critically reviewed. The majority of these techniques have been known for many years as a result of research on plasma membrane ion channels. Hence, in this review, we focus on the critical evaluation of techniques used in the studies of mitochondrial potassium channels, describing their advantages and limitations. Functional analysis of mitochondrial potassium channels in comparison to that of plasmalemmal channels presents additional experimental challenges. The reliability of functional studies of mitochondrial potassium channels is often affected by the need to isolate mitochondria and by functional properties of mitochondria such as respiration, metabolic activity, swelling capacity, or high electrical potential. Three types of techniques are critically evaluated: electrophysiological techniques, potassium flux measurements, and biochemical techniques related to potassium flux measurements. Finally, new possible approaches to the study of the function of mitochondrial potassium channels are presented. We hope that this review will assist researchers in selecting reliable methods for studying, e.g., the effects of drugs on mitochondrial potassium channel function. Additionally, this review should aid in the critical evaluation of the results reported in various articles on mitochondrial potassium channels.
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Almohammedi, A., S. M. Kapetanaki, B. R. Wood, E. L. Raven, N. M. Storey, and A. J. Hudson. "Spectroscopic analysis of myoglobin and cytochrome c dynamics in isolated cardiomyocytes during hypoxia and reoxygenation." Journal of The Royal Society Interface 12, no. 105 (April 2015): 20141339. http://dx.doi.org/10.1098/rsif.2014.1339.
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Raman microspectroscopy was applied to monitor the intracellular redox state of myoglobin and cytochrome c from isolated adult rat cardiomyocytes during hypoxia and reoxygenation. The nitrite reductase activity of myoglobin leads to the production of nitric oxide in cells under hypoxic conditions, which is linked to the inhibition of mitochondrial respiration. In this work, the subsequent reoxygenation of cells after hypoxia is shown to lead to increased levels of oxygen-bound myoglobin relative to the initial levels observed under normoxic conditions. Increased levels of reduced cytochrome c in ex vivo cells are also observed during hypoxia and reoxygenation by Raman microspectroscopy. The cellular response to reoxygenation differed dramatically depending on the method used in the preceding step to create hypoxic conditions in the cell suspension, where a chemical agent, sodium dithionite, leads to reduction of cytochromes in addition to removal of dissolved oxygen, and bubbling-N 2 gas leads to displacement of dissolved oxygen only. These results have an impact on the assessment of experimental simulations of hypoxia in cells. The spectroscopic technique employed in this work will be used in the future as an analytical method to monitor the effects of varying levels of oxygen and nutrients supplied to cardiomyocytes during either the preconditioning of cells or the reperfusion of ischaemic tissue.
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Costa, Alexandre D. T., Casey L. Quinlan, Anastasia Andrukhiv, Ian C. West, Martin Jabůrek, and Keith D. Garlid. "The direct physiological effects of mitoKATP opening on heart mitochondria." American Journal of Physiology-Heart and Circulatory Physiology 290, no. 1 (January 2006): H406—H415. http://dx.doi.org/10.1152/ajpheart.00794.2005.
Full textAbstract:
The mitochondrial ATP-sensitive K+ channel (mitoKATP) has been assigned multiple roles in cell physiology and in cardioprotection. Each of these roles must arise from basic consequences of mitoKATP opening that should be observable at the level of the mitochondrion. MitoKATP opening has been proposed to have three direct effects on mitochondrial physiology: an increase in steady-state matrix volume, respiratory stimulation (uncoupling), and matrix alkalinization. Here, we examine the evidence for these hypotheses through experiments on isolated rat heart mitochondria. Using perturbation techniques, we show that matrix volume is the consequence of a steady-state balance between K+ influx, caused either by mitoKATP opening or valinomycin, and K+ efflux caused by the mitochondrial K+/H+ antiporter. We show that increasing K+ influx with valinomycin uncouples respiration like a classical uncoupler with the important difference that uncoupling via K+ cycling soon causes rupture of the outer mitochondrial membrane and release of cytochrome c. By loading the potassium binding fluorescent indicator into the matrix, we show directly that K+ influx is increased by diazoxide and inhibited by ATP and 5-HD. By loading the fluorescent probe BCECF into the matrix, we show directly that increasing K+ influx with either valinomycin or diazoxide causes matrix alkalinization. Finally, by comparing the effects of mitoKATP openers and blockers with those of valinomycin, we show that four independent assays of mitoKATP activity yield quantitatively identical results for mitoKATP-mediated K+ transport. These results provide decisive support for the hypothesis that mitochondria contain an ATP-sensitive K+ channel and establish the physiological consequences of mitoKATP opening for mitochondria.
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Urban, Otmar, Petr Holub, and Karel Klem. "Seasonal courses of photosynthetic parameters in sun- and shade-acclimated spruce shoots." Beskydy 10, no. 1-2 (2017): 49–56. http://dx.doi.org/10.11118/beskyd201710010049.
Full textAbstract:
Exponential attenuation of light intensity passing through forest canopies leads to the formation of sun- and shade-acclimated leaves contributing to overall canopy carbon gain. Using a gas-exchange technique, seasonal changes in photosynthetic parameters were investigated in situ to test the hypothesis that the relative contributions of sun- and shade-acclimated Norway spruce shoots to total carbon gain vary during the growing season and that the contribution of sun-acclimated shoots to total carbon uptake may be reduced during the hot and dry summer season. In agreement with the tested hypothesis, we found reduced photosynthetic capacity as well as reduced light-use efficiency for carbon assimilation in sun-acclimated shoots during summer months while these remained almost unchanged in shade-acclimated shoots. Reduction of photosynthetic capacity was primarily associated with reduced stomatal conductance. On the other hand, seasonal courses of mitochondrial dark respiration, quantum efficiency of photosynthetic reactions, and compensation irradiance were primarily driven by changes in temperature. Accordingly, the photosynthetic characteristics of sun- and shade-acclimated shoots tended to converge in early spring and late autumn when temperature was low. Such seasonal dynamics result in an increased contribution of shade-acclimated shoots to total carbon uptake at the beginning and end of the growing season as well as during hot and dry summer periods.
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Agil, Ahmad, Miguel Navarro-Alarcon, Fatma Abo Zakaib Ali, Ashraf Albrakati, Diego Salagre, Cristina Campoy, and Ehab Kotb Elmahallawy. "Melatonin Enhances the Mitochondrial Functionality of Brown Adipose Tissue in Obese—Diabetic Rats." Antioxidants 10, no. 9 (September 17, 2021): 1482. http://dx.doi.org/10.3390/antiox10091482.
Full textAbstract:
Developing novel drugs/targets remains a major effort toward controlling obesity-related type 2 diabetes (diabesity). Melatonin controls obesity and improves glucose homeostasis in rodents, mainly via the thermogenic effects of increasing the amount of brown adipose tissue (BAT) and increases in mitochondrial mass, amount of UCP1 protein, and thermogenic capacity. Importantly, mitochondria are widely known as a therapeutic target of melatonin; however, direct evidence of melatonin on the function of mitochondria from BAT and the mechanistic pathways underlying these effects remains lacking. This study investigated the effects of melatonin on mitochondrial functions in BAT of Zücker diabetic fatty (ZDF) rats, which are considered a model of obesity-related type 2 diabetes mellitus (T2DM). At five weeks of age, Zücker lean (ZL) and ZDF rats were subdivided into two groups, consisting of control and treated with oral melatonin for six weeks. Mitochondria were isolated from BAT of animals from both groups, using subcellular fractionation techniques, followed by measurement of several mitochondrial parameters, including respiratory control ratio (RCR), phosphorylation coefficient (ADP/O ratio), ATP production, level of mitochondrial nitrites, superoxide dismutase activity, and alteration in the mitochondrial permeability transition pore (mPTP). Interestingly, melatonin increased RCR in mitochondria from brown fat of both ZL and ZDF rats through the reduction of the proton leak component of respiration (state 4). In addition, melatonin improved the ADP/O ratio in obese rats and augmented ATP production in lean rats. Further, melatonin reduced mitochondrial nitrosative and oxidative status by decreasing nitrite levels and increasing superoxide dismutase activity in both groups, as well as inhibited mPTP in mitochondria isolated from brown fat. Taken together, the present data revealed that chronic oral administration of melatonin improved mitochondrial respiration in brown adipocytes, while decreasing oxidative and nitrosative stress and susceptibility of adipocytes to apoptosis in ZDF rats, suggesting a beneficial use in the treatment of diabesity. Further research regarding the molecular mechanisms underlying the effects of melatonin on diabesity is warranted.
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Zhang, Qi, Lina Han, Ce Shi, Rongqing Pan, Man Chun John MA, Jeremy Ryan, Stephen E. Kurtz, et al. "Upregulation of MAPK/MCL-1 Maintaining Mitochondrial Oxidative Phosphorylation Confers Acquired Resistance to BCL-2 Inhibitor Venetoclax in AML." Blood 128, no. 22 (December 2, 2016): 101. http://dx.doi.org/10.1182/blood.v128.22.101.101.
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Abstract ABT-199 (venetoclax), a selective small-molecule antagonist of the anti-apoptotic protein BCL-2, enables the activation of pro-apoptotic proteins and the induction of cancer cell death. Our previous studies found that AML is a BCL-2 dependent disease and responds robustly to venetoclax by induction of apoptotic cell death (Pan et al., Cancer Discovery 2014). Despite initial responses to single agent venetoclax in a Phase II trial of relapsed AML, patients ultimately developed resistance and progressed (Konopleva et al., Cancer Discovery 2016). In this study we investigated mechanisms of acquired resistance to venetoclax in preclinical AML models. First, we generated 5 AML cell lines resistant to ³1µM venetoclax. No BAX (exon5 and 6) or BCL2 (exon2) mutations were found in resistant cells. Immunoblotting analysis demonstrated increased expression of anti-apoptotic proteins MCL-1, BCL-2 A1, and BCL-XL, and a decrease of pro-apoptotic PUMA protein in selected resistant cell lines. To probe the functional interactions between the pro- and anti-apoptotic proteins, we next performed co-immunoprecipitation (co-IP) studies. The anti-BIM and anti-MCL-1 co-IPs revealed reduced levels of BIM:BCL-2 complexes and increased BIM:MCL-1 complexes in resistant cells compared to their parental counterparts (Fig 1B). The BH3 profiling technique examines mitochondrial sensitivity to different BH3 mimetic peptides, and has proven to be a useful tool to determine cell dependence on anti-apoptotic BCL-2 family proteins. BH3 profiling demonstrated that resistant cells had increased responses to NOXA, MS1 and HRK peptides, indicating increased dependence on MCL-1 and/or BCL-XL (Fig 1C). To characterize the functional role of MCL-1 in resistance to venetoclax, we co-treated parental and resistant cells with selective BCL-XL or MCL-1 inhibitors A-1155463 (Leverson et al. Science Transl Med 2015) and A-1210477 (Leverson et al., Cell Death Dis 2015). The combination of venetoclax with either A-1155463 or A-1210477 showed synergistic growth inhibition in all 5 parental cell lines. Notably, 4 of the 5 resistant cell lines (OCI-AML2, Kasumi, MV4-11, MOLM13) became more sensitive to an MCL-1 inhibitor but not to a BCL-XL inhibitor (Fig 1E). However, no further sensitization was seen in combination with venetoclax in resistant cells. To characterize additional mechanisms of resistance to venetoclax in AML cells, we conducted RNA sequencing of single cell clones (2 clones/cell line) isolated from paired isogenic cells (OCI-AML2, MV4-11, MOLM13). Analysis of RNA expression patterns by gene set enrichment analysis (GSEA) revealed elevated expression of genes in the RAS/MAPK pathway (Fig 1F), consistent with increased p-ERK and p-p90-RSK protein levels (Fig 1G). Inhibition of MAPK with MEK inhibitor GDC-0973 reduced MCL-1 expression in parental but not in resistant cells, indicating that MAPK activation partially contributed to high MCL-1 levels (Fig 1G). GSEA of RNAseq data further uncovered altered expression of genes involved in mitochondrial oxidative phosphorylation (OxPhos) in 3 resistant cell lines with high MCL-1 expression (OCI-AML2, MV4-11 and MOLM-13). Notably, BCL-2 was reported to sustain AML stem cell survival through maintenance of the mitochondrial activity of OxPhos (Lagadinou etal., Cell Stem Cell, 2013). Analysis of mitochondrial respiration using a Seahorse Bioanalyzer demonstrated similar levels of oxygen consumption rate (OCR) in parental and resistant cells. Inhibition of BCL-2 with 100nM venetoclax for only 2 hrs. fully blocked baseline and maximal respiratory activity in parental but not in resistant cells. In turn, inhibition of MCL-1 with A-1210477 inhibited respiration in both parental and resistant cells, indicating a role for MCL-1 in sustaining mitochondrial activity in venetoclax-resistant AML cells, which can maintain unperturbed mitochondrial function. In summary, we identified a novel mechanism of resistance to targeted BCL-2 inhibition through upregulation of MAPK leading to increased levels of anti-apoptotic MCL-1 that binds and neutralizes BIM and maintains the mitochondrial OxPhos pathway in AML cells. Concomitant inhibition of BCL-2 and MCL-1, or of BCL-2 and OxPhos could induce synergistic cell death in AML and conceivably prevent the emergence of venetoclax resistance. Disclosures Tyner: Constellation Pharmaceuticals: Research Funding; Janssen Research & Development: Research Funding; Agios Pharmaceuticals: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding; Inctye: Research Funding; Seattle Genetics: Research Funding; Aptose Biosciences: Research Funding; AstraZeneca: Research Funding; Takeda Pharmaceuticals: Research Funding; Leap Oncology: Consultancy. Leverson:AbbVie: Employment, Other: Shareholder in AbbVie. Letai:Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.
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Wu, Qiwei, Hsiang-i. Tsai, Haitao Zhu, and Dongqing Wang. "The Entanglement between Mitochondrial DNA and Tumor Metastasis." Cancers 14, no. 8 (April 7, 2022): 1862. http://dx.doi.org/10.3390/cancers14081862.
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Mitochondrial DNA, the genetic material in mitochondria, encodes essential oxidative phosphorylation proteins and plays an important role in mitochondrial respiration and energy transfer. With the development of genome sequencing and the emergence of novel in vivo modeling techniques, the role of mtDNA in cancer biology is gaining more attention. Abnormalities of mtDNA result in not only mitochondrial dysfunction of the the cancer cells and malignant behaviors, but regulation of the tumor microenvironment, which becomes more aggressive. Here, we review the recent progress in the regulation of cancer metastasis using mtDNA and the underlying mechanisms, which may identify opportunities for finding novel cancer prediction and therapeutic targets.
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Elander, Anna, Michael Sjöström, Fredrik Lundgren, Tore Scherstén, and Ann-Christin Bylund-Fellenius. "Biochemical and Morphometric Properties of Mitochondrial Populations in Human Muscle Fibres." Clinical Science 69, no. 2 (August 1, 1985): 153–63. http://dx.doi.org/10.1042/cs0690153.
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1. Two mitochondrial subpopulations were evaluated with biochemical and morphological techniques in human gastrocnemius muscle of 10 patients with peripheral arterial insufficiency and 12 control individuals. 2. The subsarcolemmal mitochondria were released by gentle homogenization, with a recovery of 32-37%, and the intermyofibrillar by enzymic digestion and further mechanical disintegration, recovery 18-21%. The subsarcolemmal mitochondria were morphologically defined as those located within 2 μm from the sarcolemma membrane and the intermyofibrillar mitochondria as those located in the rest of the fibre. 3. In the controls the intermyofibrillar mitochondria had a lower respiratory ratio than the subsarcolemmal, owing to a higher state II respiration. The subsarcolemmal space, which contained 25% of the mitochondria, had a mitochondrial volume density two- to three-fold that of the intermyofibrillar space in the controls. 4. The patients, who had a 48-64% higher oxidative enzyme capacity in their muscle tissue, had higher respiratory rate and respiratory control index with similar ADP/O ratio in the subsarcolemmal fraction in comparison with the controls. The citrate synthase activity was higher in both mitochondrial fractions of the patients. The volume densities of mitochondria, total as well as for both subpopulations, were also higher in the patients, which was further reflected in higher yields of mitochondrial protein. 5. The results demonstrate that both sub-populations of muscle mitochondria are able to adapt quantitatively and/or qualitatively. Furthermore, they show that the increased oxidative enzyme capacity of the patients is associated with an increased quantity of both mitochondrial populations and a qualitative improvement of the respiratory activity of the subsarcolemmal mitochondria.
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Di Bartolomeo, Francesca, Carl Malina, Kate Campbell, Maurizio Mormino, Johannes Fuchs, Egor Vorontsov, Claes M. Gustafsson, and Jens Nielsen. "Absolute yeast mitochondrial proteome quantification reveals trade-off between biosynthesis and energy generation during diauxic shift." Proceedings of the National Academy of Sciences 117, no. 13 (March 17, 2020): 7524–35. http://dx.doi.org/10.1073/pnas.1918216117.
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Saccharomyces cerevisiae constitutes a popular eukaryal model for research on mitochondrial physiology. Being Crabtree-positive, this yeast has evolved the ability to ferment glucose to ethanol and respire ethanol once glucose is consumed. Its transition phase from fermentative to respiratory metabolism, known as the diauxic shift, is reflected by dramatic rearrangements of mitochondrial function and structure. To date, the metabolic adaptations that occur during the diauxic shift have not been fully characterized at the organelle level. In this study, the absolute proteome of mitochondria was quantified alongside precise parametrization of biophysical properties associated with the mitochondrial network using state-of-the-art optical-imaging techniques. This allowed the determination of absolute protein abundances at a subcellular level. By tracking the transformation of mitochondrial mass and volume, alongside changes in the absolute mitochondrial proteome allocation, we could quantify how mitochondria balance their dual role as a biosynthetic hub as well as a center for cellular respiration. Furthermore, our findings suggest that in the transition from a fermentative to a respiratory metabolism, the diauxic shift represents the stage where major structural and functional reorganizations in mitochondrial metabolism occur. This metabolic transition, initiated at the mitochondria level, is then extended to the rest of the yeast cell.
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Burwell, Lindsay S., Sergiy M. Nadtochiy, Andrew J. Tompkins, Sara Young, and Paul S. Brookes. "Direct evidence for S-nitrosation of mitochondrial complex I." Biochemical Journal 394, no. 3 (February 24, 2006): 627–34. http://dx.doi.org/10.1042/bj20051435.
Full textAbstract:
NO• (nitric oxide) is a pleiotropic signalling molecule, with many of its effects on cell function being elicited at the level of the mitochondrion. In addition to the well-characterized binding of NO• to the CuB/haem-a3 site in mitochondrial complex IV, it has been proposed by several laboratories that complex I can be inhibited by S-nitrosation of a cysteine. However, direct molecular evidence for this is lacking. In this investigation we have combined separation techniques for complex I (blue-native gel electrophoresis, Superose 6 column chromatography) with sensitive detection methods for S-nitrosothiols (chemiluminescence, biotin-switch assay), to show that the 75 kDa subunit of complex I is S-nitrosated in mitochondria treated with S-nitrosoglutathione (10 μM–1 mM). The stoichiometry of S-nitrosation was 7:1 (i.e. 7 mol of S-nitrosothiols per mol of complex I) and this resulted in significant inhibition of the complex. Furthermore, S-nitrosothiols were detected in mitochondria isolated from hearts subjected to ischaemic preconditioning. The implications of these results for the physiological regulation of respiration, for reactive oxygen species generation and for a potential role of S-nitrosation in cardioprotection are discussed.
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Lai, Raymond E., Matthew E. Holman, Qun Chen, Jeannie Rivers, Edward J. Lesnefsky, and Ashraf S. Gorgey. "Assessment of mitochondrial respiratory capacity using minimally invasive and noninvasive techniques in persons with spinal cord injury." PLOS ONE 17, no. 3 (March 11, 2022): e0265141. http://dx.doi.org/10.1371/journal.pone.0265141.
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Purpose Muscle biopsies are the gold standard to assess mitochondrial respiration; however, biopsies are not always a feasible approach in persons with spinal cord injury (SCI). Peripheral blood mononuclear cells (PBMCs) and near-infrared spectroscopy (NIRS) may alternatively be predictive of mitochondrial respiration. The purpose of the study was to evaluate whether mitochondrial respiration of PBMCs and NIRS are predictive of respiration of permeabilized muscle fibers after SCI. Methods Twenty-two individuals with chronic complete and incomplete motor SCI between 18–65 years old were recruited to participate in the current trial. Using high-resolution respirometry, mitochondrial respiratory capacity was measured for PBMCs and muscle fibers of the vastus lateralis oxidizing complex I, II, and IV substrates. NIRS was used to assess mitochondrial capacity of the vastus lateralis with serial cuff occlusions and electrical stimulation. Results Positive relationships were observed between PBMC and permeabilized muscle fibers for mitochondrial complex IV (r = 0.86, P < 0.0001). Bland-Altman displayed agreement for complex IV (MD = 0.18, LOA = -0.86 to 1.21), between PBMCs and permeabilized muscles fibers. No significant relationships were observed between NIRS mitochondrial capacity and respiration in permeabilized muscle fibers. Conclusions This is the first study to explore and support the agreement of less invasive clinical techniques for assessing mitochondrial respiratory capacity in individuals with SCI. The findings will assist in the application of PBMCs as a viable alternative for assessing mitochondrial health in persons with SCI in future clinical studies.
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Callahan, Leigh A., and Gerald S. Supinski. "Diaphragm and cardiac mitochondrial creatine kinases are impaired in sepsis." Journal of Applied Physiology 102, no. 1 (January 2007): 44–53. http://dx.doi.org/10.1152/japplphysiol.01204.2005.
Full textAbstract:
Previous studies indicate that ATP formation by the electron transport chain is impaired in sepsis. However, it is not known whether sepsis affects the mitochondrial ATP transport system. We hypothesized that sepsis inactivates the mitochondrial creatine kinase (MtCK)-high energy phosphate transport system. To examine this issue, we assessed the effects of endotoxin administration on mitochondrial membrane-bound creatine kinase, an important trans-mitochondrial ATP transport system. Diaphragms and hearts were isolated from control ( n = 12) and endotoxin-treated (8 mg·kg−1·day−1; n = 13) rats after pentobarbital anesthesia. We isolated mitochondria using techniques that allow evaluation of the functional coupling of mitochondrial creatine kinase MtCK activity to oxidative phosphorylation. MtCK functional activity was established by 1) determining ATP/creatine-stimulated oxygen consumption and 2) assessing total creatine kinase activity in mitochondria using an enzyme-linked assay. We examined MtCK protein content using Western blots. Endotoxin markedly reduced diaphragm and cardiac MtCK activity, as determined both by ATP/creatine-stimulated oxygen consumption and by the enzyme-linked assay (e.g., ATP/creatine-stimulated mitochondrial respiration was 173.8 ± 7.3, 60.5 ± 9.3, 210.7 ± 18.9, was 67.9 ± 7.3 natoms O·min−1·mg−1 in diaphragm control, diaphragm septic, cardiac control, and cardiac septic samples, respectively; P < 0.001 for each tissue comparison). Endotoxin also reduced diaphragm and cardiac MtCK protein levels (e.g., protein levels declined by 39.5% in diaphragm mitochondria and by 44.2% in cardiac mitochondria; P < 0.001 and P = 0.009, respectively, comparing sepsis to control conditions). Our data indicate that endotoxin markedly impairs the MtCK-ATP transporter system; this phenomenon may have significant effects on diaphragm and cardiac function.
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Shiota, M., Y. Fujimoto, M. Inagami, M. Hiramatsu, M. Moriyama, K. Kimura, M. Ohta, and T. Sugano. "Adaptive changes in zonation for gluconeogenic capacity in liver lobules of cold-exposed rats." American Journal of Physiology-Endocrinology and Metabolism 265, no. 4 (October 1, 1993): E559—E564. http://dx.doi.org/10.1152/ajpendo.1993.265.4.e559.
Full textAbstract:
The rate of gluconeogenesis from lactate increased in perfused livers after exposure of rats to cold for 5 days, and it returned to the control rate after 20 days [M. Shiota, T. Tanaka, and T. Sugano. Am. J. Physiol. 249 (Endocrinol. Metab. 12): E281-E286, 1985.]. The relationship between the increased gluconeogenic activity and its zonal distribution in liver lobules was studied in cold-exposed rats that had been starved for 24 h by examination of preparations enriched for periportal hepatocytes (PP-H) and for perivenous hepatocytes (PV-H), which had been isolated by the digitonin-collagenase perfusion technique. In the control group, the rate of gluconeogenesis from lactate or alanine was three times higher in PP-H than in PV-H. The rate of gluconeogenesis from these substrates in PP-H was not changed by exposure of rats to cold. The rates of PV-H increased to the level in PP-H after 5 days of exposure of rats to cold and then returned to the control rates after 20 days. The rate of gluconeogenesis from fructose was not altered in either preparation of cells by cold treatment of rats. The change in gluconeogenic capacity in PV-H caused by exposure of rats to cold was unrelated to changes in the activity of the malate-aspartate shuttle and of pyruvate kinase. The increased capacity in mitochondrial respiration was observed in both preparations of cells by cold treatment of rats for 5 days. The activity of phosphoenolpyruvate carboxykinase was higher in PP-H than in PV-H in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)
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Rich, P. R. "The molecular machinery of Keilin's respiratory chain." Biochemical Society Transactions 31, no. 6 (December 1, 2003): 1095–105. http://dx.doi.org/10.1042/bst0311095.
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Keilin's classic paper of 1925 [Keilin (1925) Proc. R. Soc. London Ser. B 100, 129–151], achieved with simple, but elegant, techniques, describes the cytochrome components of the respiratory chain and their roles in intracellular respiration and oxygen consumption. Since that time, a tremendous amount of work has clarified the intricate details of the prosthetic groups, cofactors and proteins that comprise the respiratory chain and associated machinery for ATP synthesis. The work has culminated in advanced crystallographic and spectroscopic methods that provide structural and mechanistic details of this mitochondrial molecular machinery, in many instances to atomic level. I review here the current state of understanding of the mitochondrial respiratory chain in terms of structures and dynamics of the component proteins and their roles in the biological electron and proton transfer processes that result in ATP synthesis. These advances, together with emerging evidence of further diverse roles of mitochondria in health and disease, have prompted a new era of interest in mitochondrial function.
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Vielhaber, S., A. Kudin, R. Schröder, C. E. Elger, and W. S. Kunz. "Muscle fibres: applications for the study of the metabolic consequences of enzyme deficiencies in skeletal muscle." Biochemical Society Transactions 28, no. 2 (February 1, 2000): 159–64. http://dx.doi.org/10.1042/bst0280159.
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Mitochondrial function in saponin-permeabilized muscle fibres can be studied by high-resolution respirometry, laser-excited fluorescence spectroscopy and fluorescence microscopy. We applied these techniques to study metabolic effects of changes in the pattern of mitochondrial enzymes in skeletal muscle of patients with chronic progressive external ophthalmoplegia or Kearns-Sayre syndrome harbouring large-scale deletions of mitchondrial DNA (mtDNA). In all patients combined deficiencies of respiratory chain enzymes containing mitochondrially encoded subunits were observed. The citrate synthase-normalized activity ratios of these enzymes decreased linearly with increasing mtDNA heteroplasmy. This indicates the absence of any well-defined mutation thresholds for mitochondrial enzyme activities in the entire skeletal muscle. We applied metabolic control analysis to perform a quantitative estimation of the metabolic influence of the observed enzyme deficiencies. For patients with degrees of mtDNA heteroplasmy below about 60% we observed at almost normal maximal rates of respiration an increase in flux control coefficients of complexes I and IV. Permeabilized skeletal-muscle fibres of patients with higher degrees of mtDNA heteroplasmy and severe enzyme deficiencies exhibited additionally decreased maximal rates of respiration. This finding indicates the presence of a ‘metabolic threshold’ which can be assessed by functional studies of muscle fibres providing the link to the phenotypic expression of the mtDNA mutation in skeletal muscle.
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Politis-Barber, Valerie, Henver S. Brunetta, Sabina Paglialunga, Heather L. Petrick, and Graham P. Holloway. "Long-term, high-fat feeding exacerbates short-term increases in adipose mitochondrial reactive oxygen species, without impairing mitochondrial respiration." American Journal of Physiology-Endocrinology and Metabolism 319, no. 2 (August 1, 2020): E376—E387. http://dx.doi.org/10.1152/ajpendo.00028.2020.
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White adipose tissue (WAT) dysfunction in obesity is implicated in the onset of whole body insulin resistance. Alterations in mitochondrial bioenergetics, namely impaired mitochondrial respiration and increased mitochondrial reactive oxygen species (mtROS) production, have been suggested to contribute to this metabolic dysregulation. However, techniques investigating mitochondrial function are classically normalized to tissue weight, which may be confounding when considering obesity-related adipocyte hypertrophy. Furthermore, the effect of long-term high-fat diet (HFD) on mtROS in WAT has yet to be elucidated. Therefore, we sought to determine the HFD-mediated temporal changes in mitochondrial respiration and mtROS emission in WAT. C57BL/6N mice received low-fat diet or HFD for 1 or 8 wk and changes in inguinal WAT (iWAT) and epididymal WAT (eWAT) were assessed. While tissue weight-normalized mitochondrial respiration was reduced in iWAT following 8-wk HFD-feeding, this effect was mitigated when adipocyte cell size and/or number were considered. These data suggest HFD does not impair mitochondrial respiratory capacity per adipocyte within WAT. In support of this assertion, within eWAT compensatory increases in lipid-supported and maximal succinate-supported respiration occurred at 8 wk despite cell hypertrophy and increases in WAT inflammation. Although these data suggest impairments in mitochondrial respiration do not contribute to HFD-mediated WAT phenotype, lipid-supported mtROS emission increased following 1-wk HFD in eWAT, while both lipid and carbohydrate-supported mtROS were increased at 8 wk in both depots. Combined, these data establish that while HFD does not impair adipocyte mitochondrial respiratory capacity, increased mtROS is an enduring physiological occurrence within WAT in HFD-induced obesity.
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Serudji, Joserizal, Nuzulia Irawati, Johanes Cornelius Mose, Hirowati Ali, and Yusrawati Yusrawati. "Serum HIF-1α Levels, miR-210 Expressions, and Reactive Oxygen Species Levels in Early Abortion and Normal Pregnancy." Open Access Macedonian Journal of Medical Sciences 10, B (August 4, 2022): 1779–83. http://dx.doi.org/10.3889/oamjms.2022.9325.
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Background: The blastocyst implants in a relatively hypoxic state. Hypoxic state triggers hypoxia-inducible factor-1α (HIF-1ɑ) production, upregulates the transcription factor miR-210, and stimulates reactive oxygen species (ROS) production by trophoblast cells. HIF-1α also increases the expression of miR-210. High expression of micro-RNA 210 (miR-210) suppresses mitochondrial respiration, increasing ROS production. High level of ROS may result in DNA damage or cell disfunction, thereby impaired trophoblast invasion, leading to early abortion. This study aims to determine the differences of serum HIF-1ɑ levels, miR-210 expressions, and ROS levels between early abortion and normal pregnancy.
Method: This cross-sectional comparative study was conducted in Dr. M. Djamil Hospital Padang, Andalas University Hospital, and 5 Public Health Centers in Padang. Fifty-patients with gestational age less than 12 weeks (25 early abortions and 25 normal pregnancies) were included in this study. All samples were tested for HIF-1ɑ and ROS level using enzyme-linked immunosorbent assay (ELISA) method, and miR-210 expression using real-time polymerase chain reaction (PCR) technique. Spearman correlation and Mann Whitney test. was used in this study.
Results: Both study groups were equivalent in terms of age, gestational age, and gravidity (p = 0.51, 0.453 and 1.00). The median of HIF-1ɑ level, miR-210 expression, and ROS level were higher in early abortions than normal pregnancies i.e (3.73 vs 3.42) ng/mL (p = 0.016), (43.55 vs 17.85) copies/ng (p = 0.027), and (1.36 vs 1.20) ng/mL (p = 0.003). The coefficient correlations were 0.16 between HIF-1ɑ level and miR-210 expression (p=0.267), 0.46 between HIF-1ɑ level and ROS level (p=0.001), and 0.18 between miR-210 expression and ROS level (p=0.207).
Conclusion: HIF-1ɑ level, miR-210 expression, and ROS level were associated with early abortion. HIF-1ɑ level has a correlation with ROS level.
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Firth, Amy L., Kathryn H. Yuill, and Sergey V. Smirnov. "Mitochondria-dependent regulation of Kv currents in rat pulmonary artery smooth muscle cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 295, no. 1 (July 2008): L61—L70. http://dx.doi.org/10.1152/ajplung.90243.2008.
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Voltage-gated K+ (Kv) channels are important in the regulation of pulmonary vascular function having both physiological and pathophysiological implications. The pulmonary vasculature is essential for reoxygenation of the blood, supplying oxygen for cellular respiration. Mitochondria have been proposed as the major oxygen-sensing organelles in the pulmonary vasculature. Using electrophysiological techniques and immunofluorescence, an interaction of the mitochondria with Kv channels was investigated. Inhibitors, blocking the mitochondrial electron transport chain at different complexes, were shown to have a dual effect on Kv currents in freshly isolated rat pulmonary arterial smooth muscle cells (PASMCs). These dual effects comprised an enhancement of Kv current in a negative potential range (manifested as a 5- to 14-mV shift in the Kv activation to more negative membrane voltages) with a decrease in current amplitude at positive potentials. Such effects were most prominent as a result of inhibition of Complex III by antimycin A. Investigation of the mechanism of antimycin A-mediated effects on Kv channel currents ( IKv) revealed the presence of a mitochondria-mediated Mg2+ and ATP-dependent regulation of Kv channels in PASMCs, which exists in addition to that currently proposed to be caused by changes in intracellular reactive oxygen species.
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Viengchareun, Say, Martine Caron, Martine Auclair, Min Ji Kim, Paule Frachon, Jacqueline Capeau, Marc Lombès, and Anne Lombès. "Mitochondrial Toxicity of Indinavir, Stavudine and Zidovudine Involves Multiple Cellular Targets in white and brown adipocytes." Antiviral Therapy 12, no. 6 (August 2007): 919–30. http://dx.doi.org/10.1177/135965350701200610.
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Objective To evaluate the mechanisms of mitochondrial toxicity associated with antiretroviral treatment. Methods 3T3–F442A white and T37i brown adipocytes were exposed to stavudine (10 μM), zidovudine (1 μM) and indinavir (10 μM), alone or in combination. Adipocyte fat content was measured with Oil Red O staining. Quantification of mRNA levels and of mitochondrial DNA content used PCR-based techniques. Mitochondrial activities were evaluated with respiration, ATP synthesis and spectrophotometric assays. Mitochondrial mass was assessed by the fluorescent probe MitoTracker Red. Results In both cell types, all the treatments induced a severe defect of adipogenesis (low lipid content and decreased markers of adipogenic maturation: peroxisome proliferator-activated receptor [PPAR]γ2 and aP2 but also uncoupling protein 1 in brown adipocytes) as well as altered mitochondrial function (decreased respiration rate and increased mitochondrial mass). Drug combination did not give additional toxicity. Brown adipocytes appeared more affected than white adipocytes (lower respiration rate and decreased ATP production). The mechanisms of mitochondrial toxicity differed with the drug and the cell type. Only stavudine induced severe mitochondrial DNA depletion in both cell types. With all the treatments, white adipocytes showed a decrease in the expression of mitochondrial and nuclear-DNA-encoded respiratory chain subunits (cytochrome c oxidase [CytOx]2 and CytOx4), whereas brown adipocytes maintained normal expression in accordance with their increase of the transcriptional factors of mitochondrial biogenesis nuclear respiratory factor 1 and PPARγ coactivator (PGC)1-related cofactor PRC, but not PGC1α. Conclusion Our results provide evidence for dissociation between mitochondrial activity, transcription and mitochondrial DNA content, highlighting the complexity of mitochondrial toxicity, which affects multiple cellular targets.
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Simeonidis, Evangelos, Ettore Murabito, Kieran Smallbone, and Hans V. Westerhoff. "Why does yeast ferment? A flux balance analysis study." Biochemical Society Transactions 38, no. 5 (September 24, 2010): 1225–29. http://dx.doi.org/10.1042/bst0381225.
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Advances in biological techniques have led to the availability of genome-scale metabolic reconstructions for yeast. The size and complexity of such networks impose limits on what types of analyses one can perform. Constraint-based modelling overcomes some of these restrictions by using physicochemical constraints to describe the potential behaviour of an organism. FBA (flux balance analysis) highlights flux patterns through a network that serves to achieve a particular objective and requires a minimal amount of data to make quantitative inferences about network behaviour. Even though FBA is a powerful tool for system predictions, its general formulation sometimes results in unrealistic flux patterns. A typical example is fermentation in yeast: ethanol is produced during aerobic growth in excess glucose, but this pattern is not present in a typical FBA solution. In the present paper, we examine the issue of yeast fermentation against respiration during growth. We have studied a number of hypotheses from the modelling perspective, and novel formulations of the FBA approach have been tested. By making the observation that more respiration requires the synthesis of more mitochondria, an energy cost related to mitochondrial synthesis is added to the FBA formulation. Results, although still approximate, are closer to experimental observations than earlier FBA analyses, at least on the issue of fermentation.
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Jacob, W. A., R. Hertsens, A. Van Bogaert, and M. De Smet. "Mitochondrial matrix calcium ions regulate energy production in myocardium: A cytochemical study." Proceedings, annual meeting, Electron Microscopy Society of America 48, no. 2 (August 12, 1990): 166–67. http://dx.doi.org/10.1017/s0424820100134429.
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In the past most studies of the control of energy metabolism focus on the role of the phosphorylation potential ATP/ADP.Pi on the regulation of respiration. Studies using NMR techniques have demonstrated that the concentrations of these compounds for oxidation phosphorylation do not change appreciably throughout the cardiac cycle and during increases in cardiac work. Hence regulation of energy production by calcium ions, present in the mitochondrial matrix, has been the object of a number of recent studies.Three exclusively intramitochondnal dehydrogenases are key enzymes for the regulation of oxidative metabolism. They are activated by calcium ions in the low micromolar range. Since, however, earlier estimates of the intramitochondnal calcium, based on equilibrium thermodynamic considerations, were in the millimolar range, a physiological correlation was not evident. The introduction of calcium-sensitive probes fura-2 and indo-1 made monitoring of free calcium during changing energy metabolism possible. These studies were performed on isolated mitochondria and extrapolation to the in vivo situation is more or less speculative.
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Marzano, Giuseppina, Natalina Moscatelli, Mariangela Di Giacomo, Nicola Antonio Martino, Giovanni Michele Lacalandra, Maria Elena Dell’Aquila, Giuseppe Maruccio, et al. "Centrifugation Force and Time Alter CASA Parameters and Oxidative Status of Cryopreserved Stallion Sperm." Biology 9, no. 2 (January 27, 2020): 22. http://dx.doi.org/10.3390/biology9020022.
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Conventional sperm selection techniques used in ARTs rely on centrifugation steps. To date, the different studies reported on the effects of centrifugation on stallion sperm motility provided contrasting results and do not include effects on mitochondrial functionality and different oxidative parameters. The effects of different centrifugation protocols (300× g for 5′, 300× g for 10′, 1500× g for 5′ and 1500× g for 10′ vs. no centrifugation) on motility and oxidative status in cryopreserved stallion sperm, were analyzed. After centrifugation, almost all motility parameters were significantly altered, as observed by computer-assisted sperm analysis. A polarographic assay of oxygen consumption showed a progressive decrease in mitochondria respiration from the gentlest to the strongest protocol. By laser scanning confocal microscopy, significant reduction of mitochondrial membrane potential, at any tested protocol, and time-dependent effects, at the same centrifugal force, were found. Increased DNA fragmentation index at any tested protocol and time-dependent effects at the same centrifugal force were found, whereas increased protein carbonylation was observed only at the strongest centrifugal force. These results provide more comprehensive understandings on centrifugation-induced effects on cryopreserved stallion sperm and suggest that, even at a weak force for a short time, centrifugation impairs different aspects of equine sperm metabolism and functionality.
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Lieberman, S. J., W. Wasco, J. MacLeod, P. Satir, and G. A. Orr. "Immunogold localization of the regulatory subunit of a type II cAMP-dependent protein kinase tightly associated with mammalian sperm flagella." Journal of Cell Biology 107, no. 5 (November 1, 1988): 1809–16. http://dx.doi.org/10.1083/jcb.107.5.1809.
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We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.
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Cadrin, M., M. Tolszczuk, J. Guy, G. Pelletier, K. B. Freeman, and L. J. Bukowiecki. "Immunohistochemical identification of the uncoupling protein in rat brown adipose tissue." Journal of Histochemistry & Cytochemistry 33, no. 2 (February 1985): 150–54. http://dx.doi.org/10.1177/33.2.3881519.
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Brown adipose tissue mitochondria are characterized by the presence of an uncoupling protein that gives them an exceptional capacity for substrate-controlled respiration and thermogenesis. The specific localization of this protein in rat brown adipocytes was demonstrated using an immunohistochemical technique, the peroxidase-antiperoxidase (PAP) method. Light microscopy observations showed that serum antibodies raised against the uncoupling protein selectively reacted with multilocular brown adipocytes. No labeling could be detected in either unilocular adipocytes, capillaries, or muscle fibers (striated and vascular smooth muscle). Staining was more intensive in certain adipocytes than in others, suggesting the presence of cellular heterogeneity. The specificity of the staining technique was demonstrated by showing that treatment of the preparations with antiserum saturated with an excess of uncoupling protein almost entirely inhibited brown adipocyte labeling. The specificity and selectivity of the PAP method allow the clear differentiation of uncoupling protein-containing adipocytes from other cellular types, suggesting that this immunohistochemical technique will represent an extremely useful tool for studying adipocyte function and differentiation.
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Fuentes-Mateos, Rocío, Eugenio Santos, and Alberto Fernández-Medarde. "Optimized Protocol for Isolation and Culture of Murine Neonatal Primary Lung Fibroblasts." Methods and Protocols 6, no. 1 (January 24, 2023): 14. http://dx.doi.org/10.3390/mps6010014.
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During all the stages of lung development, the lung mesoderm (or mesenchyme) is closely related to the endoderm, and their cross-regulation promotes, controls, and drives all lung developmental processes. Generation of 3D organoids in vitro, RNA assays, and mitochondrial respiration studies are used to analyze lung development and regeneration to better understand the interactions between epithelium and mesenchyme, as well as for the study of redox alterations and the metabolic status of the cells. Moreover, to avoid using immortalized cell lines in these in vitro approaches, standardized murine neonatal primary lung fibroblast isolation techniques are essential. Here, we present an optimized method to isolate, culture, and freeze primary lung fibroblasts from neonatal lungs. Our current method includes step-by-step instructions accompanied by graphical explanations and critical steps.
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Lemar, Katey M., Ourania Passa, Miguel A. Aon, Sonia Cortassa, Carsten T. Müller, Sue Plummer, Brian O'Rourke, and David Lloyd. "Allyl alcohol and garlic (Allium sativum) extract produce oxidative stress in Candida albicans." Microbiology 151, no. 10 (October 1, 2005): 3257–65. http://dx.doi.org/10.1099/mic.0.28095-0.
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Both the growth and respiration of Candida albicans are sensitive to extracts of Allium sativum and investigations into the anticandidal activities are now focussing on the purified constituents to determine the targets of inhibition. Of particular interest is allyl alcohol (AA), a metabolic product that accumulates after trituration of garlic cloves. Putative targets for AA were investigated by monitoring changes in intracellular responses after exposure of C. albicans cells to AA or a commercially available garlic extract. Two-photon laser scanning microscopy and other techniques were used. Changes typical of oxidative stress – NADH oxidation and glutathione depletion, and increased reactive oxygen species – were observed microscopically and by flow cytometry. Known targets for AA are alcohol dehydrogenases Adh1 and 2 (in the cytosol) and Adh3 (mitochondrial), although the significant decrease in NAD(P)H after addition of AA is indicative of another mechanism of action.