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A V Kuznetsov

Publications and source records attributed to A V Kuznetsov.

At least 55 records · Page 3Linked to original sources
PubMed1994

Actions of the creatine analogue beta-guanidinopropionic acid on rat heart mitochondria.

The action of the creatine analogue beta-guanidinopropionic acid (beta-GPA) was examined in rat heart mitochondria and in isolated cardiomyocytes or fibres which were permeabilized with the non-ionic detergent saponin to determine the kinetics of mitochondrial creatine kinase for beta-GPA. Fibres and myocytes were subjected to increasing [ADP] in the presence and absence of beta-GPA or creatine, whereas isolated mitochondria received a similar protocol with increasing [ATP]. In isolated mitochondria given ATP, there was a stimulation of respiration by creatine, but no significant stimulation of respiration by beta-GPA. Further studies on fibres from control and beta-GPA-fed rats also found that beta-GPA is not utilized by the mitochondria, as evidenced by a lack of beta-GPA-stimulated respiration (Km for ADP = 142 +/- 23 microM) compared with control (Km for ADP from 161 +/- 23 microM), but no significant change in Vmax. Therefore the rat heart mitochondria are not responsive to beta-GPA as compared with creatine. Interestingly, the fibres from beta-GPA-fed rats had no creatine- or beta-GPA-stimulated respiration (Km for ADP = 57.3 +/- 7.2 microM for control, 54.2 +/- 7.2 microM with creatine, and 53.5 +/- 7.8 microM with beta-GPA). The mitochondria prepared from the hearts of rats exposed for 10 weeks to 1% beta-GPA in their diet had a significant decrease in Vmax. and a significant decrease in Km for ADP. Thus the hearts from beta-GPA-fed animals may be pathologic, due to a disruption of the creatine kinase energy circuit.

Adenosine Triphosphate↗
PubMed1994

Measurement of fluorescence changes of NAD(P)H and of fluorescent flavoproteins in saponin-skinned human skeletal muscle fibers.

Saponin-skinned human muscle fibers from M. vastus lateralis were immobilized in a quartz capillary to detect the fluorescence changes of NAD(P)H and of fluorescent flavoproteins. To get sufficient intense fluorescence signals from a small amount of muscle tissue the NAD(P)H fluorescence was excited by means of an HeCd laser at 325 nm and the flavoprotein fluorescence by an argon-ion laser at 454 nm or by the second wavelength of a HeCd laser at 442 nm. Using this experimental setup the fluorescence spectra of NAD(P)H, of alpha-lipoamide dehydrogenase and of electron-transfer flavoprotein were detected in saponin-skinned human muscle fibers. These fibers behaved identically to isolated mitochondria: (i) The addition of substrates caused an increase in reduction of mitochondrial NAD+, (ii) the addition of ADP caused its reoxidation, and (iii) the addition of respiratory chain inhibitors led to an almost complete reduction of NAD+. It was observed that the redox state of the NAD(P) system and of the alpha-lipoamide dehydrogenase reached after addition of 1 mM ADP correlates with the rate of active state respiration with NAD-dependent substrates. Therefore, this fluorimetric method is suitable to compare the mitochondrial oxidation capacities of NAD-dependent substrates in less then 5 mg wet weight muscle tissue. Moreover, the maximal changes in fluorescence of NAD(P)H and flavoproteins correlate with the amount of mitochondrial marker enzymes per milligram muscle tissue. Using this method a myopathy caused by a diminished content of mitochondria per milligram muscle tissue was observed.

Adenosine Diphosphate↗
PubMed1993

Retarded diffusion of ADP in cardiomyocytes: possible role of mitochondrial outer membrane and creatine kinase in cellular regulation of oxidative phosphorylation.

Possible reasons for retarded intracellular diffusion of ADP were investigated. The isolated skinned cardiac fibers were used to study apparent kinetic parameters for externally added ADP in control of mitochondrial respiration. Participation of myosin-ATPase in binding of ADP within cells as it was supposed earlier (Saks, V.A., Belikova, Yu.O. and Kuznetsov, A.V. (1991) Biochim. Biophys. Acta 1074, 302-311) was completely excluded, since myosin-deprived skinned cardiac fibers ('ghosts') displayed the same kinetic parameters as intact ones (Kmapp for ADP about 300 microM). Significantly lower apparent Km values were obtained for fibers with osmotically disrupted outer mitochondrial membrane (25-35 microM), which was close to that observed for isolated heart mitochondria. The data obtained are in favor of limitation of ADP movement via anion-selective low-conductance porine channels in the outer membrane of mitochondria. It is proposed that the permeability of this membrane is controlled by some unknown intracellular factor(s). In the presence of saturating concentrations of creatine (25 mM) the apparent Km for ADP significantly decreases due to coupling of creatine kinase and oxidative phosphorylation reactions in mitochondria. This coupling is not observed in KCl medium in which mitochondrial creatine kinase is detached from the membrane. It is concluded that in the cells in-vivo ADP movement between cytoplasm and intramitochondrial space is controlled by low-conductivity anion channels in the outer membrane. Thus, the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocase is an important mechanism in control of oxidative phosphorylation in vivo due to its ability to manifold amplify these very weak ADP signals from cytoplasm.

Adenosine Diphosphate↗
PubMed1993

Functional characterization of mitochondrial oxidative phosphorylation in saponin-skinned human muscle fibers.

The conditions of treatment of human skeletal muscle fibers from M. vastus lateralis with saponin were optimized to achieve complete permeabilization of cell membrane at intact mitochondrial oxidative phosphorylation. After 30 min of incubation with saponin all lactate dehydrogenase, 50% of creatine kinase, 30% of adenylate kinase and less than 20% of citrate synthase was released into the permeabilization medium. These skinned fibers behave similar to isolated mitochondria from human skeletal muscle: (i) the respiration with mitochondrial substrates can be stimulated by ADP, (ii) inhibited by carboxyatractyloside and (iii) it is possible to detect fluorescence changes of mitochondrial NAD(P)H on additions of substrates, uncoupler and cyanide. From a comparison of rates of respiration per cytochrome aa3 content of isolated human skeletal muscle mitochondria and saponin-skinned muscle fibers it was possible to calculate that almost 85% of mitochondria in those fibers are accessible for the investigation of oxidative phosphorylation. As shown by the investigation of biopsy samples of two patients with undefined myopathies these fibers are a suitable object for the replacement of isolated mitochondria in the diagnosis of mitochondrial myopathies and encephalomyopathies.

Animals↗
PubMed1993

Mitochondrial oxidative phosphorylation in saponin-skinned human muscle fibers is stimulated by caffeine.

The addition of 15 mM caffeine to saponin-skinned human muscle fibers from M. vastus lateralis caused in the presence of 2 mM ATP an approx. 2-fold stimulation of respiration with glutamate+malate. This effect can be abolished by either the addition of the Ca2+ chelator EGTA, the inhibitor of Ca2+ transport Ruthenium red and the inhibitor of the myosin ATPase vanadate. The caffeine concentration dependency of respiration of fibers coincided with the caffeine-caused stimulation of myosin ATPase activity. The activation of oxidative phosphorylation in saponin-skinned human muscle fibers by caffeine can be explained by a stimulation of myosin ATPase caused by Ca2+ release from sarcoplasmic reticulum.

Caffeine↗
PubMed1993

Influence of the mitochondrial outer membrane and the binding of creatine kinase to the mitochondrial inner membrane on the compartmentation of adenine nucleotides in the intermembrane space of rat heart mitochondria.

The influence of the mitochondrial outer membrane and that of the binding of creatine kinase to the mitochondrial inner membrane on the compartmentation of adenine nucleotides in the intermembrane space of rat heart mitochondria were investigated under conditions of maximal rates of mitochondrial creatine kinase. To this end, experiments were performed in reconstituted systems consisting of functionally intact rat heart mitochondria or mitoplasts and pyruvate kinase, both competing at ADP formed by mitochondrial creatine kinase in the presence of high concentrations of adenine nucleotides. Results showed that removal of the mitochondrial outer membrane diminished the compartmentation effects, supporting the relevance of the mitochondrial outer membrane to compartmentation effects in the intermembrane space. Compartmentation effects were clearly seen even in KCl-containing media which release the creatine kinase from the inner membrane, indicating that the localization of the ADP-regenerating enzyme in the mitochondrial intermembrane space within the outer membrane is a prerequisite for dynamic compartmentation of adenine nucleotides. Under these conditions, the binding of creatine kinase to the inner membrane is of minor importance. Lowering the ATP concentrations diminished the extent of AdN compartmentation due to decreased creatine kinase rates. Under these conditions of low AdN fluxes, the release of creatine kinase from the inner membrane entailed another decrease in the compartmentation.

Adenine Nucleotides↗
PubMed1993

[Mott cells in the lymph].

For the first time Mott cells have been found in the lymph. The lymph was obtained with the help of glass micropipettes from cisterna chyli of rabbits. The Mott cells in Romanovsky-Giemsa-stained lymph smears were examined in a light microscope. The Mott cells of the lymph preserve their unique morphological characteristics. They usually have a rounded form. The cytoplasm of Mott cells is divided into many liquid spheres which dominate the cell interior and are grouped in several rows round a blue-stained nucleus. The number of the Mott cells in the lymph doubles in atherosclerosis in comparison with the norm. It is related with the reaction of the immune system to atherosclerosis.

Animals↗
PubMed1993

[A new method for sampling lymph from animals].

In experiments conducted on locally anesthesized rats, an access to the cistern of the thoracic duct was made by the previously developed method which involved laparatomy, the exfoliation of the left kidney, arteria renalis, vena renalis, vasa lymphatica, renal lymph nodes, and ureter, followed by drawing them medially. The cistern of the thoracic duct was punctured by glass micropipettes manufactured by the Russia's patent No. 1495076, which allowed dendritic and Mott cells to be detected in central lymph.

Anesthesia, Local↗
PubMed1993

[The possible role of the inner mitochondrial membrane in regulating oxidative phosphorylation in cells in vivo].

It has been shown for the first time that the outer mitochondrial membrane has a low permeability for ADP and can control its diffusion into cells in vivo. Respiration of saponin-skinned cardiac and skeletal muscle fibers is maximally stimulated by millimolar concentrations of external ADP. The apparent Km values for ADP are equal to 297 +/- 35 and 334 +/- 54 microM, respectively. After complete extraction of myosin with 0.8 M KCl, which fully preserves the intact structure of the mitochondria, the apparent Km values for exogenously added ADP does not change. However, disruption of the outer mitochondrial membrane by osmotic shock (treatment with 40 mOsM KCl) causes a reduction of the apparent Km value down to 32.3 +/- 5.0 microM of ADP. The apparent Km for ADP in isolated heart mitochondria is 17.6 +/- 1.0 microM. It is concluded that there exists an intracellular factor in the cells in vivo which controls the outer mitochondrial membrane and notably decreases its permeability for ADP. After isolation of mitochondria this factor is lost. When mitochondrial creatine kinase is activated, weak intracellular fluxes of ADP passing through the outer mitochondrial membrane in the skinned fibers are amplified manifold due to the tight functional coupling between mitochondrial creatine kinase and the oxidative phosphorylation system. This coupling is considered to be the central mechanism in the control of cell respiration.

Adenosine Diphosphate↗
PubMed1993

[Effect of training on the structural-metabolic indicators in athletes' skeletal muscles].

Muscle biopsy samples were taken from m.vastus lateralis of ski-runners during the training season and 4 months after training (consisting presumably of aerobic exercises). Metabolic enzyme activities, fiber cross-sectional areas, capillary supply indices and parameters of oxygen uptake by intact mitochondria in skinned fibers were measured. Training did not induce any appreciable changes in the NADH-tetrazolium reductase activity, maximal oxygen uptake by mitochondria, mean fiber size or capillary density. At the same time, there were significant increases in the cytochrome c oxidase activity, respiratory control index and creatine-stimulated respiratory rate. The activities of alpha-glycerophosphate dehydrogenase, malate dehydrogenase and myoglobin peroxidase as well as the basal respiratory rate of mitochondria decreased as a result of training. The data obtained testify to the possibility of qualitative adaptations in the respiratory chain of mitochondria.

Adolescent↗
PubMed1992

The creatine kinase system and cardiomyopathy.

Changes in the creatine kinase system, cellular energetics, regulation of respiration and alterations in parameters of contractility in experimental animals (myopathic hamsters), and in patients with dilated cardiomyopathy were studied. 31P-NMR methods were used to show that cardiomyopathic hearts are characterized by decreased work index, lower tissue ATP, phosphocreatine, and total creatine contents and diminished creatine kinase activity and energy fluxes. In isolated mitochondria, only the creatine kinase activity was decreased. Both in cardiomyopathic hamsters and human hearts a share of mitochondrial creatine kinase in the total tissue enzyme activity was decreased from 33% to 18% and that of BB elevated from 5% in control to 20%, at an unchanged relative level of MM. In saponins-skinned cardiac fibers on cardiomyocytes creatine (Cr, 25 mM) decreased Km for ADP in regulation of respiration from 133 +/- 20 to 20 +/- 4 microM due to activation of coupled mitochondrial creatine kinase-oxidative phosphorylation reactions in control hamster hearts. In the case of cardiomyopathy it decreased Km for ADP only to 81 +/- 13 microM. In endocardial biopsy samples from the hearts of patients with dilated cardiomyopathy taken during angiography, creatine stimulated respiration was decreased by 36% of control value, which correlated well with increase of end-diastolic pressure and fall in ejection fraction. Thus, changes in mitochondrial creatine kinase expression diminished the efficiency of cellular regulation of respiration in cardiomyopathic hearts that may have functional consequences for hemodynamics or may be adaptive alterations in response to decreased contractility.

Animals↗
PubMed1992

[Quantitative relationship between ischemic heart disease and parameters of energy metabolism].

The relationship between coronary heart disease, postischemic work recovery and tissue ATP levels as well as mitochondrial respiration rates were studied. Respiration of mitochondria was assessed without their isolation by using a novel method applying skinned fibers in physiological saline. The maximal mitochondrial respiration rates were unchanged during 35 min of normothermic ischemia in St. Thomas Hospital cardioplegic solution in the subsequent 30 min aerobic reperfusion period. A reversible increase in the basal respiration and a decrease in creatine-stimulated oxygen uptake were observed. Thus, the combined determination of mitochondrial respiration in situ in skinned cardiac fibers and tissue ATP may be a useful approach to studies of the pathogenesis of cardiac diseases.

Adenosine Triphosphate↗
PubMed1992

[Phenotypes of dendritic cells in central lymph of healthy rabbits and during correction of experimental atherosclerosis].

Dendritic cells of central lymph of rabbits have been identified according to the form of the cell body, characteristics of formation and branchiness of its processes in health, in atherosclerosis, its correction with radon, polyphenol preparations made of Sanguisorba officinalis and in combination of the latter. Two main types of dendritic cells have been distinguished. Type I is characterized by a rounded body with clear outlines, protrusions and one compact process. Such cells are often found in lymph of intact animals. Type II has a cell body of various forms with two and more compact or branching processes. This type is mainly detected in atherosclerosis and its correction. The prevalence of the above phenotypes of dendritic cells is attributed to the response of the immune system to atherosclerosis and its correction.

Animals↗
PubMed1991

In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP.

Relative diffusivities of ADP and creatine in cardiomyocytes were studied. The isolated rat cardiomyocytes were lysed with saponin (40 micrograms/ml) to perforate or completely disrupt sarcolemma that was evidenced by leakage of 80-100% lactate dehydrogenase. In these cardiomyocytes mitochondria were used as 'enzymatic probes' to determine the average local concentration of substrates exerting acceptor control of respiration--ADP or creatine (the latter activates respiration via mitochondrial creatine kinase reaction)--when their concentrations in the surrounding medium were changed. The kinetic parameters for ADP and creatine in control of respiration of saponin-treated cardiomyocytes were compared with those determined in isolated mitochondria and skinned cardiac fibers. The apparent Km for creatine (at 0.2 mM ATP) was very close and in a range of 6.0-6.9 mM in all systems studied, showing the absence of diffusion difficulties for this substrate. On the contrary, the apparent Km for ADP increased from 18 +/- 1 microM for isolated mitochondria to 250 +/- 59 microM for cardiomyocytes with the lysed sarcolemma and to 264 +/- 57 microM for skinned fibers. This elevation of Km was not eliminated by inhibition of myokinase with diadenosine pentaphosphate. When 25 mM creatine was present, the apparent Km for ADP decreased to 36 +/- 6 microM. These data are taken to indicate specific restrictions of diffusion of ADP most probably due to its interaction with intermediate binding sites in cardiomyocytes. The important role of phosphocreatine-creatine kinase system of energy transport is to overcome the restrictions in regulation of energy fluxes due to decreased diffusivity of ADP.

Adenosine Diphosphate↗
PubMed1991

Phosphocreatine pathway for energy transport: ADP diffusion and cardiomyopathy.

Chemically skinned (by treatment with saponin, 40 micrograms/ml) isolated cardiomyocytes were used to study the intracellular diffusion of ADP and creatine (Cr). Stimulation of respiration was studied in these cardiomyocytes without intact sarcolemma and in isolated heart mitochondrial by addition of ADP and Cr in the presence of 0.2 mM ATP (via mitochondrial creatine kinase reaction: Cr + MgATP = MgADP + PCr). The Michaelis constant (Km) for Cr was similar in both cases, 5.67 +/- 0.11 (SD) mM in skinned myocytes and 6.9 +/- 0.2 mM in mitochondria, showing that there is no significant restriction to the diffusion of this substrate. However, the apparent Km for external ADP increased from 17.6 +/- 1.0 microM for mitochondria to 250 +/- 38 microM for skinned cardiomyocytes, showing decreased diffusivity of ADP as a result of binding to cellular structures. In the presence of 25 mM Cr, the Km for ADP for myocytes decreased to 35.6 +/- 5.6 microM due to the coupling of the creatine kinase and oxidative phosphorylation reactions. Provision of substrate for the creatine kinase reaction amplified the weak ADP signal in the regulation of respiration. The activity of the mitochondrial creatine kinase was decreased by a factor of two in cardiomyopathic hamsters and human hearts and was associated with a twofold decrease in creatine-stimulated respiration. These data show a potentially key role of mitochondrial creatine kinase in the regulation of cellular respiration and the possible importance of changes in its activity for the functional disturbances of the cardiomyopathic heart.

Adenosine Diphosphate↗