Jose Olivares
Joseph Fourier University
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Molecular and Cellular Biochemistry | 1998
Valdur Saks; Vladimir Veksler; Andrei V. Kuznetsov; Laurence Kay; Peeter Sikk; Toomas Tiivel; Leone Tranqui; Jose Olivares; Kirstin Winkler; Falk R. Wiedemann; Wolfram S. Kunz
In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than 10 years of research in four countries is summarized. The use of saponin in very low concentration (50–100 μg/ml) for permeabilisation of the sarcolemma leaves all intracellular structures, including mitochondria, completely intact. The intactness of mitochondrial function in these skinned muscle fibers is demonstrated in this work by multiple methods, such as NADH and flavoprotein fluorescence studies, fluorescence imaging, confocal immunofluorescence microscopy and respiratory analysis. Permeabilized cell and skinned fiber techniques have several very significant advantages for studies of mitochondrial function, in comparison with the traditional methods of use of isolated mitochondria: (1) very small tissue samples are required; (2) all cellular population of mitochondria can be investigated; (3) most important, however, is that mitochondria are studied in their natural surrounding. The results of research by using this method show the existence of several new phenomenon — tissue dependence of the mechanism of regulation of mitochondrial respiration, and activation of respiration by selective proteolysis. These phenomena are explained by interaction of mitochondria with other cellular structures in vivo. The details of experimental studies with use of these techniques and problems of kinetic analysis of the results are discussed. Examples of large-scale clinical application of these methods are given. (Mol Cell Biochem 184: 81–100, 1998)
Experimental Physiology | 2003
Florence Appaix; Andrey V. Kuznetsov; Yves Usson; Laurence Kay; Tatiana Andrienko; Jose Olivares; Tuuli Kaambre; Peeter Sikk; Raimund Margreiter; Valdur Saks
The origin of significant differences between the apparent affinities of heart mitochondrial respiration for exogenous ADP in isolated mitochondria in vitro and in permeabilized cardiomyocytes or skinned fibres in situ is critically analysed. All experimental data demonstrate the importance of structural factors of intracellular arrangement of mitochondria into functional complexes with myofibrils and sarcoplasmic reticulum in oxidative muscle cells and the control of outer mitochondrial membrane permeability. It has been shown that the high apparent Km for exogenous ADP (250‐350 µM) in permeabilized cells and in ghost cells (without myosin) and fibres (diameter 15‐20 µm) is independent of intrinsic MgATPase activity. However, the Km may be decreased significantly by a selective proteolytic treatment, which also destroys the regular arrangement of mitochondria between sarcomeres and increases the accessibility of endogenous ADP to the exogenous pyruvate kinase‐phosphoenolpyruvate system. The confocal microscopy was used to study the changes in intracellular distribution of mitochondria and localization of cytoskeletal proteins, such as desmin, tubulin and plectin in permeabilized cardiac cells during short proteolytic treatment. The results show the rapid collapse of microtubular and plectin networks but not of desmin localization under these conditions. These results point to the participation of cytoskeletal proteins in the intracellular organization and control of mitochondrial function in the cells in vivo, where mitochondria are incorporated into functional complexes with sarcomeres and sarcoplasmic reticulum.
Biochimica et Biophysica Acta | 2000
Florence Appaix; Marie-Nathalie Minatchy; Catherine Riva-Lavieille; Jose Olivares; Bruno Antonsson; Valdur Saks
This paper recalls the earlier work by Keilin, Margoliash and others at the beginning of the 20th century and shows how their results can be used for the rapid solution of new problems of modern science. It describes a rapid and simple spectrophotometric method for quantitative determination of cytochrome c release from isolated mitochondria or permeabilized cells induced by proapoptotic proteins. For this, the Soret (gamma) peak at 414 nm in the spectrum of cytochrome c is used. The results of spectrophotometric assay of cytochrome c release are in accord with those of oxygraphic determination of cytochrome c-dependent respiration of isolated mitochondria and permeabilized cardiomyocytes.
Molecular and Cellular Biochemistry | 2001
Laure Menin; Marina Panchichkina; C. Keriel; Jose Olivares; Urmo Braun; Enn K. Seppert; Valdur Saks
Distribution 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 μg/ml for 30 min or 600 μg/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.
Cardiovascular Drugs and Therapy | 1998
A. Rossi; Jose Olivares
The metabolism of pyrimidine nucleotides in the myocardium is poorly understood. The turnover of these nucleotides is high, whereas their concentration is rather low. The de novo pathway of synthesis does not seem very efficient, although the utilization of nucleosides could represent the major pathway for pyrimidine nucleotide synthesis. In rat blood, cytidine could be the major precursor for pyrimidine nucleotide synthesis. The precursor, whatever its exact nature (uridine or cytidine), could be species dependent, and the liver could play a major role in providing blood nucleosides. Owing to the essential role of pyrimidine nucleotides in the synthesis of macromolecules, acute or chronic alteration of the metabolism of these nucleotides could have crucial consequences on heart viability and function. Providing pyrimidine precursors to the heart, isolated or in situ, induces functional and metabolic effects on the heart. The experimental results suggest that such interventions could be beneficial in clinical situations such as cardioplegia, heart preservation, or recovery from ischemia.
Journal of Molecular and Cellular Cardiology | 1988
Jose Olivares; A. Rossi
Plasma contains micromolar concentrations of pyrimidine bases and nucleosides: uracil 1.9 +/- 0.1 (n = 29), cytosine 1.1 +/- 0.01 (n = 29), uridine 1.2 +/- 0.1 (n = 56) and cytidine 5.4 +/- 0.1 (n = 59) (means +/- S.E.M.). Accordingly, we postulated that these compounds could be used as the main precursors for myocardial pyrimidine nucleotide synthesis. The kinetics of the incorporation of blood plasma bases and nucleosides into myocardial nucleotides was studied by in vivo radio-isotopic studies in rats. The clearance of the radiolabelled compounds in blood plasma and the incorporation of radiolabelled precursors into liver nucleotides was also investigated. The results can be summarized as follows: (1) The pyrimidine radioactive bases were only very slightly incorporated into heart nucleotides 1 h after injection: thus 1 h after injection any incorporation of radiolabelled cytosine into nucleotides remained undetectable and for radioactive uracil the ratio of the specific radioactivity of uracil nucleotides to that of plasma uracil remained below 0.7%. (2) Radiolabelled plasma uridine was subject to a far more rapid catabolism than radiolabelled plasma cytidine. (3) The labelling of myocardial uracil nucleotides from plasma uridine was very slight. Their specific radioactivity represented less than 16% of plasma uridine specific radioactivity. (4) When the tracer was cytidine the specific radioactivity of cytosine nucleotides reached that of precursor within 30 min after injection and uracil nucleotides were also labeled (10% of cytidine nucleotides specific radioactivity). These results, and other previous data, suggest the possibility that the pyrimidine nucleotide synthesis in the rat heart may be largely achieved via the phosphorylation of blood plasma cytidine.
Archive | 2003
Valdur Saks; Florence Appaix; Yves Usson; Karen Guerrero; Jose Olivares; Enn Seppet; Mayis K. Aliev; Raimund Margreiter; Andrey V. Kuznetsov
Experimental and in silico studies of regulation of mitochondrial respiration in permeabilized muscle fibers have revealed functional complexes of mitochondria with myofibrils and sarcoplasmic reticulum—intracellular energetic units—as basic pattern of organization of cell energy metabolism in oxidative muscle cells (Saks et al. Biochem. J. 356, 643–657, 2001),. In this paper we review new data showing that in the cells in vivo there are at least two major factors of the control of mitochondrial function which are lost in experiments in vitro: mitochondrial position in the cell with respect to other cellular structures, and the tightly controlled mitochondrial outer membrane. Because of this, the local concentration of ADP available in the vicinity of ATP/ADP translocase is strictly controlled by the organized metabolic networks of energy transfer including the creatine kinase and adenylate kinase systems within the intracellular energetic units. Mathematical modelling of energy fluxes within these units satisfactorily explains the linear relationship between heart work and oxygen uptake under conditions of metabolic stability, and allows to quantitatively analyse the mechanism of the regulation of mitochondrial function in the cells in vivo. The knowledge of metabolic changes within the functional complexes of mitochondria with MgATPases in normal and pathological conditions may help to explain the mechanism of acute ischemic cardiac failure.
American Journal of Physiology-cell Physiology | 2005
Marko Vendelin; Nathalie Beraud; Karen Guerrero; Tatiana Andrienko; Andrey V. Kuznetsov; Jose Olivares; Laurence Kay; Valdur Saks
Biochimica et Biophysica Acta | 1997
Laurence Kay; Zhenlin Li; Mathias Mericskay; Jose Olivares; Leone Tranqui; Eric Fontaine; Toomas Tiivel; Peeter Sikk; Tuuli Kaambre; Jeane-Lyze Samuel; L. Rappaport; Yves Usson; Xavier Leverve; Denise Paulin; Valdur Saks
Developmental Biology | 1991
Agnes Fichard; Jean-Marc Verna; Jose Olivares; Raymond Saxod