Action potentials in right and left ventricles from chronic hypoxic rats: effect of almitrine.

International audienceRats were exposed to a 3-wk regimen of chronic normobaric hypoxia, with or without almitrine treatment. Chronic hypoxia led to a significant rise in the right ventricular mass and lengthened the action potential duration (APD) in right ventricle and in nonhypertrophied left ventricle. Hypertrophy and APD lengthening were significantly enhanced by almitrine. The classical acute hypoxia-induced shortening in APD was much larger in hypoxia-adapted hearts. In these hearts, almitrine treatment almost completely prevented such shortening, while the acute hypoxia-induced decrease in cardiac contraction was similar with or without almitrine. It is concluded that APD lengthening during chronic hypoxia can occur independently of ventricular hypertrophy. The similar directional effects of almitrine and chronic hypoxia on APD support the hypothesis of an energy-linked phenomenon. The dissociation of the almitrine effect on APD and contractility is in accordance with the view of a cellular energy compartmentation

Determination of mitochondrial reactive oxygen species: methodological aspects.

International audienceThe generation of Reactive Oxygen Species (ROS) as by-products in mitochondria Electron Transport Chain (ETC) has long been admitted as the cost of aerobic energy metabolism with oxidative damages as consequence. The purpose of this methodological review is to present some of the most widespread methods of ROS generation and to underline the limitations as well as some problems, identified with some experiments as examples, in the interpretation of such results. There is now no doubt that besides their pejorative role, ROS are involved in a variety of cellular processes for the continuous adaptation of the cell to its environment. Because ROS metabolism is a complex area (low production, instability of species, efficient antioxidant defense system, several places of production...) bias, variances and limitations in ROS measurements must be recognized in order to avoid artefactual conclusions, and especially to improve our understanding of physiological and pathophysiological mechanisms of such phenomenon

Mechanism of gluconeogenesis inhibition in rat hepatocytes isolated after in vivo hypoxia.

International audienceGluconeogenesis was studied in hepatocytes isolated from fasted rats submitted to 24 h of hypoxic exposure (inspired O2 fraction 0.1) or to room air. Hepatocytes from hypoxic rats compared with controls exhibited a lower gluconeogenic rate with lactate (5.1 +/- 0.3 vs. 7.2 +/- 0.3 mumol.min-1.g dry cells-1, P < 0.001) but not with dihydroxyacetone (9.1 +/- 0.3 vs. 9.4 +/- 0.4 mumol.min-1.g dry cells-1), suggesting involvement of the phosphoenolpyruvate-pyruvate cycle. Experiments with perifused hepatocytes from hypoxic and control rats showed a single relationship between phosphoenolpyruvate and glucose flux (JGlc) but two different curves when cytosolic oxalacetate was plotted against JGlc. The decreased phosphoenolpyruvate carboxykinase (PEPCK) activity in the hypoxic group (9.0 +/- 0.9 vs. 16.2 +/- 1.9 nmol.min-1.mg protein-1, P < 001) without change in the Michaelis constant further settled the involvement of this step. The significant decrease in PEPCK mRNA levels in livers from hypoxic rats led us to propose that in vivo hypoxic exposure inhibits gluconeogenesis at the PEPCK level by decreasing PEPCK gene transcription

Macrocompartmentation of total creatine in cardiomyocytes revisited.

International audienceDistribution of total creatine (free creatine + phosphocreatine) between two subcellular macrocompartments--mitochondrial matrix space and cytoplasm--in heart and skeletal muscle cells was reinvestigated by using a permeabilized cell technique. Isolated cardiomyocytes were treated with saponin (50 microg/ml for 30 min or 600 microg/ml for 1 min) to open the outer cellular membrane and release the metabolites from cytoplasm (cytoplasmic fraction, CF). All mitochondrial population in permeabilized cells remained intact: the outer membrane was impermeable for exogenous cytochrome c, the acceptor control index of respiration exceeded 10, the mitochondrial creatine kinase reaction was fully coupled to the adenine nucleotide translocator. Metabolites were released from mitochondrial fraction (MF) by 2-5% Triton X100. Total cellular pool of free creatine + phosphocreatine (69.6 +/- 2.1 nmoles per mg of protein) was found exclusively in CF and was practically absent in MF. When fibers were prepared from perfused rat hearts, cellular distribution of creatine was not dependent on functional state of the heart and only slightly modified by ischemia. It is concluded that there is no stable pool of creatine or phosphocreatine in the mitochondrial matrix in the intact muscle cells, and the total creatine pool is localized in only one macrocompartment--cytoplasm

Octanoate affects 2,4-dinitrophenol uncoupling in intact isolated rat hepatocytes.

International audienceWhen intact isolated rat hepatocytes, either incubated or perifused, were uncoupled by 2,4-dinitrophenol, we found that the effect on glucose and lactate+pyruvate fluxes, cytosolic and mitochondrial redox states and ATP/ADP ratios were dependent on the nature of the exogenous substrate added. 2,4-Dinitrophenol addition (0.25 mmol/l) to cells perifused with dihydroxyacetone (10 mmol/l) resulted in a modest and transient activation of oxygen uptake accompanied by a surprising rise in lactate/pyruvate ratio indicating an increase in the cytosolic NADH/NAD+ ratio. In addition, such uncoupling, fully abolished glucose production, enhanced lactate+pyruvate flux, and strongly decreased cytosolic and mitochondrial ATP/ADP ratios. In these steady-state conditions, further addition of octanoate (0.4 mmol/l) induced a large and sustained enhancement of respiration with a concomitant decrease in the lactate/pyruvate ratio, whereas glucose flux was restored to some extent and cytosolic and mitochondrial ATP/ADP ratios increased. Inhibition of the malate-aspartate shuttle by the transaminase inhibitor aminooxyacetate (0.3 mmol/l) did not modify the effect of 2,4-dinitrophenol with dihydroxyacetone alone whereas it decreased the maximal stimulation of oxygen uptake after octanoate addition. In view of these results we propose the following conclusions. The uncoupling of intact cells by 2,4-dinitrophenol inhibits the translocation of reducing equivalents into the mitochondrial matrix probably by impairing the malate-aspartate shuttle. This explains the increase in the cytosolic NADH/NAD+ ratio and the transient activation of respiration with dihydroxyacetone. Fatty acid addition to cells uncoupled with 2,4-dinitrophenol appears to restore a mitochondrial membrane potential, probably by providing the respiratory chain with reduced cofactors directly in the matrix, thus allowing the transfer of reducing equivalents across the mitochondrial membrane. The restoration, to some extent, of a protonmotive force to uncoupled cells by fatty acid addition is also supported by an increase in ATP synthesis as evidenced by a glucose synthesis with dihydroxyacetone as gluconeogenic substrate

Cytoplasmic cellular structures control permeability of outer mitochondrial membrane for ADP and oxidative phosphorylation in rat liver cells.

International audienceThe kinetics of regulation mitochondrial respiration by external ADP in permeabilized hepatocytes was studied further. In digitonin-permeabilized hepatocytes, the apparent Km for ADP in regulation of respiration was decreased from 275 +/- 35 microM in control to 48 +/- 8 microM by a treatment with trypsin (15 min, 0.125 mg/ml). In liver tissue homogenates, trypsin treatment similarly decreased the Km value for ADP. These results show that ADP diffusion in hepatocytes may be retarded due to some unknown cytoplasmic trypsin-sensitive protein factor(s) which may be lost during isolation of mitochondria. Since we have previously reported a limited permeability of the outer mitochondrial membrane in isolated hepatocytes (Saks et al. 1995, Biochem. Biophys. Res. Commun., 208, 919-926), we conclude that an important site of control of respiration in liver cells in vivo is located at the porin channels of the outer mitochondrial membrane

Est-il possible, en médecine nucléaire, d'étudier le métabolisme cardiaque par détection externe ?

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Correlation between degree of rupture of outer mitochondrial membrane and changes of kinetics of regulation of respiration by ADP in permeabilized heart and liver cells.

International audienceKinetics of regulation of respiration by ADP in skinned cardiac fibers and permeabilized isolated hepatocytes was compared before and after mitochondrial swelling due to hypoosmotic treatment in 30-50 mOsM solutions. In both systems the apparent Km for ADP was high before mitochondrial swelling and equal to 297 +/- 35 microM and 275 +/- 35 microM, correspondingly. Hypoosmotic treatment resulted in the rupture of outer mitochondrial membrane and in the loss of exogenous cytochrome c, and both in skinned cardiac fibers and permeabilized hepatocytes the value of apparent Km for ADP was decreased in correlation with the extent of the rupture of the outer mitochondrial membrane. It is concluded that both in heart and liver cells in vivo, the ADP diffusion in the cells is retarded due to very low permeability of the outer mitochondrial membrane for ADP

Influence of the presence of a methyl group on the myocardial metabolism of 15-(paraiodophenyl)-3 methyl pentadecanoic acid (IMPPA).

International audienceThe objective of the present study was to determine the mechanism of accumulation of myocardial activity following i.v. injection of 15-(paraiodophenyl)-3 methyl pentadecanoic acid (IMPPA). IMPPA and 15 phenyl-3 methyl pentadecanoic acid (MPPA) were labeled with 14C at position 1 and used to perfuse isolated rat hearts in a closed system. After 5 min of perfusion, IMPPA reached 2/3 of its value at 45 min. 14CO2 production was low. Most of the myocardial activity was in the form of free IMPPA. Analysis of IMPPA activation by CoA SH revealed that it was very strongly inhibited. The retention of myocardial activity is thus due to intracellular accumulation of free IMPPA following inhibition of activation. Comparison of results obtained with IMPPA and MPPA showed that the presence of iodine in the molecule accentuates the inhibition of activation