Differential requirements of calcium for oxoglutarate dehydrogenase and mitochondrial nitric-oxide synthase under hypoxia: Impact on the regulation of mitochondrial oxygen consumption (original) (raw)
Related papers
Suppression of mitochondrial respiratory function after short-term anoxia
The American journal of physiology, 1987
Exposure of rat hepatocytes to 30 min anoxia resulted in a substantial decrease in O2 consumption on reoxygenation. Measurement of the sequestered Ca2+ pool of mitochondria by selective release with the protonophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and quantitation with the metallochromic indicator, arsenazo III, showed that anoxia caused a marked decrease in mitochondrial Ca2+. This loss could, in part, be due to decreased electrophoretic uptake resulting from a 20% decrease in the magnitude of the mitochondrial transmembranal potential. The decrease was associated with a decrease in ATP synthase activity as expected from the Ca2+ dependence of endogenous inhibitor binding to the ATP synthase. These results show that short-term anoxia suppresses mitochondrial function in hepatocytes and suggest that mitochondrial Ca2+ content may be important in this regulation. Regulation of the ATP synthase and other ion transport systems may provide a means to preserve ...
Redox Report, 2011
We have used two different probes with distinct detection properties, dichlorodihydrofluorescein diacetate and Amplex Red/horseradish peroxidase, as well as different respiratory substrates and electron transport chain inhibitors, to characterize the reactive oxygen species (ROS) generation by the respiratory chain in calcium-overloaded mitochondria. Regardless of the respiratory substrate, calcium stimulated the mitochondrial generation of ROS, which were released at both the mitochondrial-matrix side and the extramitochondrial space, in a way insensitive to the mitochondrial permeability transition pores inhibitor cyclosporine A. In glutamate/malate-energized mitochondria, inhibition at complex I or complex III (ubiquinone cycle) similarly modulated ROS generation at either mitochondrial-matrix side or extramitochondrial space; this also occurred when the backflow of electrons to complex I in succinateenergized mitochondria was inhibited. On the other hand, in succinate-energized mitochondria the modulation of ROS generation at mitochondrial-matrix side or extra-mitochondrial space depends on the site of complex III which was inhibited. These results allow a straight comparison between the effects of different respiratory substrates and electron transport chain inhibitors on ROS generation at either mitochondrial-matrix side or extra-mitochondrial space in calcium-overloaded mitochondria.
Calcium Overload and Mitochondrial Metabolism
Biomolecules
Mitochondria calcium is a double-edged sword. While low levels of calcium are essential to maintain optimal rates of ATP production, extreme levels of calcium overcoming the mitochondrial calcium retention capacity leads to loss of mitochondrial function. In moderate amounts, however, ATP synthesis rates are inhibited in a calcium-titratable manner. While the consequences of extreme calcium overload are well-known, the effects on mitochondrial function in the moderately loaded range remain enigmatic. These observations are associated with changes in the mitochondria ultrastructure and cristae network. The present mini review/perspective follows up on previous studies using well-established cryo–electron microscopy and poses an explanation for the observable depressed ATP synthesis rates in mitochondria during calcium-overloaded states. The results presented herein suggest that the inhibition of oxidative phosphorylation is not caused by a direct decoupling of energy metabolism via t...
Experimental Cell Research, 2007
Energy-producing pathways, adenine nucleotide levels, oxidative stress response and Ca 2+ homeostasis were investigated in cybrid cells incorporating two pathogenic mitochondrial DNA point mutations, 3243A N G and 3302A N G in tRNA Leu(UUR) , as well as Rho 0 cells and compared to their parental 143B osteosarcoma cell line. All cells suffering from a severe respiratory chain deficiency were able to proliferate as fast as controls. The major defect in oxidative phosphorylation was efficiently compensated by a rise in anaerobic glycolysis, so that the total ATP production rate was preserved. This enhancement of glycolysis was enabled by a considerable decrease of cellular total adenine nucleotide pools and a concomitant shift in the AMP + ADP/ATP ratios, while the energy charge potential was still in the normal range. Further important consequences were an increased production of superoxide which, however, was neither escorted by major changes in the antioxidative
Journal of Molecular and Cellular Cardiology, 2007
The objective of the present study was to delineate the molecular mechanisms for mitochondrial contribution to oxidative stress induced by hypoxia and reoxygenation in the heart. The present study introduces a novel model allowing real-time studying mitochondria under hypoxia and reoxygenation, and describes the significance of intramitochondrial calcium homeostasis and mitochondrial nitric oxide synthase (mtNOS) for oxidative stress. The present study shows that incubating isolated rat heart mitochondria under hypoxia followed by reoxygenation, but not hypoxia per se, causes cytochrome c release from the mitochondria, oxidative modification of mitochondrial lipids and proteins, and inactivation of mitochondrial enzymes susceptible to inactivation by peroxynitrite. Those alterations were prevented when mtNOS was inhibited or mitochondria were supplemented with antioxidant peroxynitrite scavengers. The present study shows mitochondria independent of other cellular components respond to hypoxia/reoxygenation by elevating intramitochondrial ionized calcium and stimulating mtNOS. The present study proposes a crucial role for heart mitochondrial calcium homeostasis and mtNOS in oxidative stress induced by hypoxia/ reoxygenation.
Oxygen dependence of mitochondrial nitric oxide synthase activity
Biochemical and Biophysical Research Communications, 2003
The effect of O 2 concentration on mitochondrial nitric oxide synthase (mtNOS) activity and on O À 2 production was determined in rat liver, brain, and kidney submitochondrial membranes. The K m O 2 for mtNOS were 40, 73, and 37 lM O 2 and the V max were 0.51, 0.49, and 0.42 nmol NO/min mg protein for liver, brain, and kidney mitochondria, respectively. The rates of O À 2 production, 0.5-12.8 nmol O À 2 /min mg protein, depended on O 2 concentration up to 1.1 mM O 2 . Intramitochondrial NO, O À 2 , and ONOO À steady-state concentrations were calculated for the physiological level of 20 lM O 2 ; they were 20-39 nM NO, 0.17-0.33 pM O À 2 , and 0.6-2.2 nM ONOO À for the three organs. These levels establish O 2 /NO ratios of 513-1000 that correspond to physiological inhibitions of cytochrome oxidase by intramitochondrial NO of 16-25%. The production of NO by mtNOS appears as a regulatory process that modulates mitochondrial oxygen uptake and cellular energy production.
Journal of Biological Chemistry, 2011
Mitochondrial TCA cycle dehydrogenase enzymes have been shown to be stimulated by Ca 2؉ under various substrate and ADP incubation conditions in an attempt to determine and understand the role of Ca 2؉ in maintaining energy homeostasis in working hearts. In this study, we tested the hypothesis that, at physiological temperature and 1 mM extramitochondrial free magnesium, Ca 2؉ can stimulate the overall mitochondrial NAD(P)H generation flux in rat heart mitochondria utilizing pyruvate and malate as substrates at both subsaturating and saturating concentrations. In both cases, we found that, in the physiological regime of mitochondrial oxygen consumption observed in the intact animal and in the physiological range of cytosolic Ca 2؉ concentration averaged per beat, Ca 2؉ had no observable stimulatory effect. A modest apparent stimulatory effect (22-27%) was observable at supraphysiological maximal ADP-stimulated respiration at 2.5 mM initial phosphate. The stimulatory effects observed over the physiological Ca 2؉ range are not sufficient to make a significant contribution to the control of oxidative phosphorylation in the heart in vivo.
Open-Loop Control of Oxidative Phosphorylation in Skeletal and Cardiac Muscle Mitochondria by Ca2+
Biophysical Journal, 2016
In cardiac muscle, mitochondrial ATP synthesis is driven by demand for ATP through feedback from the products of ATP hydrolysis. However, in skeletal muscle at higher workloads there is an apparent contribution of open-loop stimulation of ATP synthesis. Open-loop control is defined as modulation of flux through a biochemical pathway by a moiety, which is not a reactant or a product of the biochemical reactions in the pathway. The role of calcium, which is known to stimulate the activity of mitochondrial dehydrogenases, as an open-loop controller, was investigated in isolated cardiac and skeletal muscle mitochondria. The kinetics of NADH synthesis and respiration, feedback from ATP hydrolysis products, and stimulation by calcium were characterized in isolated mitochondria to test the hypothesis that calcium has a stimulatory role in skeletal muscle mitochondria not apparent in cardiac mitochondria. A range of respiratory states were obtained in cardiac and skeletal muscle mitochondria utilizing physiologically relevant concentrations of pyruvate and malate, and flux of respiration, NAD(P)H fluorescence, and rhodamine 123 fluorescence were measured over a range of extra mitochondrial calcium concentrations. We found that under these conditions calcium stimulates NADH synthesis in skeletal muscle mitochondria but not in cardiac mitochondria.