RESUMO
Physiological role of creatine (Cr) became first evident in the experiments of Belitzer and Tsybakova in 1939, who showed that oxygen consumption in a well-washed skeletal muscle homogenate increases strongly in the presence of creatine and with this results in phosphocreatine (PCr) production with PCr/O(2) ratio of about 5-6. This was the beginning of quantitative analysis in bioenergetics. It was also observed in many physiological experiments that the contractile force changes in parallel with the alteration in the PCr content. On the other hand, it was shown that when heart function is governed by Frank-Starling law, work performance and oxygen consumption rate increase in parallel without any changes in PCr and ATP tissue contents (metabolic homeostasis). Studies of cellular mechanisms of all these important phenomena helped in shaping new approach to bioenergetics, Molecular System Bioenergetics, a part of Systems Biology. This approach takes into consideration intracellular interactions that lead to novel mechanisms of regulation of energy fluxes. In particular, interactions between mitochondria and cytoskeleton resulting in selective restriction of permeability of outer mitochondrial membrane anion channel (VDAC) for adenine nucleotides and thus their recycling in mitochondria coupled to effective synthesis of PCr by mitochondrial creatine kinase, MtCK. Therefore, Cr concentration and the PCr/Cr ratio became important kinetic parameters in the regulation of respiration and energy fluxes in muscle cells. Decrease in the intracellular contents of Cr and PCr results in a hypodynamic state of muscle and muscle pathology. Many experimental studies have revealed that PCr may play two important roles in the regulation of muscle energetics: first by maintaining local ATP pools via compartmentalized creatine kinase reactions, and secondly by stabilizing cellular membranes due to electrostatic interactions with phospholipids. The second mechanism decreases the production of lysophosphoglycerides in hypoxic heart, protects the cardiac cells sarcolemma against ischemic damage, decreases the frequency of arrhythmias and increases the post-ischemic recovery of contractile function. PCr is used as a pharmacological product Neoton in cardiac surgery as one of the components of cardioplegic solutions for protection of the heart against intraoperational injury and injected intravenously in acute myocardial ischemic conditions for improving the hemodynamic response and clinical conditions of patients with heart failure.
Assuntos
Creatina Quinase/metabolismo , Creatina/metabolismo , Metabolismo Energético , Miócitos Cardíacos/metabolismo , Fosfocreatina/metabolismo , Animais , Humanos , Miócitos Cardíacos/química , Miócitos Cardíacos/enzimologiaRESUMO
Hydrolysis of the emulsified mixture of short-chain triacylglycerols by porcine pancreatic lipase in the presence of procolipase and micellar sodium taurodeoxycholate has been studied. Increase in the content of tributyrin and trioctanoin in the mixture with triacetin had highly cooperative effects on the formation of the interfacial lipase procolipase complex. Abrupt enhancement of the complex stability was observed in the presence of 0.4-0.6 mol mol-1 of tributyrin or 0.58 mol mol-1 of trioctanoin in the substrate phase. The affinity of lipase towards interfacially bound procolipase for the trioctanoin containing 0.07-0.42 mol mol-1 of triacetin was approximately three times higher than that for pure trioctanoin. The cooperative processes involved in complex formation did not contribute to the affinity of the interfacial lipase/(pro)colipase complex towards substrate molecules and its catalytic activity.
Assuntos
Colipases/química , Lipase/química , Pâncreas/enzimologia , Triglicerídeos/química , Animais , Colipases/isolamento & purificação , Emulsões , Precursores Enzimáticos , Hidrólise , Lipase/isolamento & purificação , Precursores de Proteínas/isolamento & purificação , Solubilidade , SuínosRESUMO
Regulation of mitochondrial respiration in situ in the muscle cells was studied by using fully permeabilized muscle fibers and cardiomyocytes. The results show that the kinetics of regulation of mitochondrial respiration in situ by exogenous ADP are very different from the kinetics of its regulation by endogenous ADP. In cardiac and m. soleus fibers apparent K(m) for exogenous ADP in regulation of respiration was equal to 300-400 microM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 microM when about 70% of maximal rate of respiration was achieved. Respiration rate maintained by intracellular ATPases was suppressed about 20-30% during exogenous trapping of ADP with excess pyruvate kinase (PK, 20 IU/ml) and phosphoenolpyruvate (PEP, 5 mM). ADP flux via the external PK+PEP system was decreased by half by activation of mitochondrial oxidative phosphorylation. Creatine (20 mM) further activated the respiration in the presence of PK+PEP. It is concluded that in oxidative muscle cells mitochondria behave as if they were incorporated into functional complexes with adjacent ADP producing systems - with the MgATPases in myofibrils and Ca,MgATPases of sarcoplasmic reticulum.
Assuntos
ATPase de Ca(2+) e Mg(2+)/metabolismo , Mitocôndrias Musculares/enzimologia , Fibras Musculares Esqueléticas/enzimologia , Retículo Sarcoplasmático/enzimologia , Difosfato de Adenosina/biossíntese , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Fosfatos de Dinucleosídeos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Musculares/efeitos dos fármacos , Modelos Químicos , Miocárdio/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
The purpose of this work was to investigate the mechanism of regulation of mitochondrial respiration in vivo in different muscles of normal rat and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cells in vivo in the heart, soleus and white gastrocnemius skeletal muscles of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the "ghost" (phantom) cardiomyocytes were produced by extraction of myosin with 800 mM KCl. Use of confocal immunofluorescent microscopy and anti-desmin antibodies showed good preservation of mitochondria and cytoskeletal system in these phantom cells. Kinetics of respiration regulation by ADP was also studied in these cells in detail before and after binding of anti-desmine antibodies with intermediate filaments. In skinned cardiac or soleus skeletal muscle fibers but not in fibers from fast twitch skeletal muscle the kinetics of mitochondrial respiration regulation by ADP was characterized by very high apparent Km (low affinity) equal to 300-400 microM, exceeding that for isolated mitochondria by factor of 25. In skinned fibers from m. soleus, partial inhibition of respiration by NaN3 did not decrease the apparent Km for ADP significantly, this excluding the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative activity and by intracellular diffusion problems. However, short treatment of fibers with trypsin decreased this constant value to 40-70 microM, confirming the earlier proposition that mitochondrial sensitivity to ADP in vivo is controlled by some cytoplasmic protein. Phantom cardiomyocytes which contain mostly mitochondria and cytoskeleton and retain the normal shape, showed also high apparent Km values for ADP. Therefore, they are probably the most suitable system for studies of cellular factors which control mitochondrial function in the cells in vivo. In these phantom cells anti-desmin antibodies did not change the kinetics of respiration regulation by ADP. However, in skinned fibers from the heart and m. soleus of transgenic desmin-deficient mice some changes in kinetics of respiration regulation by ADP were observed: in these fibers two populations of mitochondria were observed, one with usually high apparent Km for ADP and the second one with very low apparent Km for ADP. Morphological observations by electron microscopy confirmed the existence of two distinct cellular populations in the muscle cells of desmin-deficient mice. The results conform to the conclusion that the reason for observed high apparent Km for ADP in regulation of oxidative phosphorylation in heart and slow twitch skeletal muscle cells in vivo is low permeability of mitochondrial outer membrane porins but not diffusion problems of ADP into and inside the cells. Most probably, in these cells there is a protein associated with cytoskeleton, which controls the permeability of the outer mitochondrial porin pores (VDAC) for ADP. Desmin itself does not display this type of control of mitochondrial porin pores, but its absence results in appearance of cells with disorganised structure and of altered mitochondrial population probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structural organisation and cytoskeleton in the cells in vivo due to the existence of still unidentified protein factor(s).
Assuntos
Difosfato de Adenosina/metabolismo , Respiração Celular/fisiologia , Citoesqueleto/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Musculares/metabolismo , Porinas , Animais , Anticorpos/imunologia , Células Cultivadas , Creatina/farmacologia , Citoesqueleto/ultraestrutura , Desmina/genética , Desmina/fisiologia , Difusão , Cinética , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Oxigênio/metabolismo , Permeabilidade , Ratos , Ratos Wistar , Azida Sódica/farmacologia , Tripsina/metabolismo , Tripsina/farmacologia , Canais de Ânion Dependentes de VoltagemRESUMO
To meet high cellular demands, the energy metabolism of cardiac muscles is organized by precise and coordinated functioning of intracellular energetic units (ICEUs). ICEUs represent structural and functional modules integrating multiple fluxes at sites of ATP generation in mitochondria and ATP utilization by myofibrillar, sarcoplasmic reticulum and sarcolemma ion-pump ATPases. The role of ICEUs is to enhance the efficiency of vectorial intracellular energy transfer and fine tuning of oxidative ATP synthesis maintaining stable metabolite levels to adjust to intracellular energy needs through the dynamic system of compartmentalized phosphoryl transfer networks. One of the key elements in regulation of energy flux distribution and feedback communication is the selective permeability of mitochondrial outer membrane (MOM) which represents a bottleneck in adenine nucleotide and other energy metabolite transfer and microcompartmentalization. Based on the experimental and theoretical (mathematical modelling) arguments, we describe regulation of mitochondrial ATP synthesis within ICEUs allowing heart workload to be linearly correlated with oxygen consumption ensuring conditions of metabolic stability, signal communication and synchronization. Particular attention was paid to the structure-function relationship in the development of ICEU, and the role of mitochondria interaction with cytoskeletal proteins, like tubulin, in the regulation of MOM permeability in response to energy metabolic signals providing regulation of mitochondrial respiration. Emphasis was given to the importance of creatine metabolism for the cardiac energy homoeostasis.
Assuntos
Respiração Celular/fisiologia , Metabolismo Energético/fisiologia , Coração/fisiologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Humanos , Consumo de Oxigênio/fisiologiaRESUMO
We carried out a combined study of UV-VUV luminescence and resonant x-ray emission from BeO single crystals with incident photon energies in the vicinity of the Be 1s absorption edge. The x-ray emission spectra show that at the Be 1s photoabsorption edge the lattice relaxation processes in the excitation site take place already on the timescale of the radiative decay of the core excitation. Comparison of the x-ray emission and the luminescence spectra indicates that the maximum energy loss of the process of lattice relaxation during the decay of inner-shell holes is similar to the loss that occurs in the self-trapping process of valence excitons. The possible decay channels of core excitations have been discussed and the mechanism for the creation of 5.2 eV luminescence at the photoabsorption resonances has been suggested.
Assuntos
Berílio/química , Óxidos/química , Teoria Quântica , Análise Espectral/métodos , Raios Ultravioleta , Difração de Raios X/métodos , Absorção , Elétrons , Transferência de Energia , Luminescência , FótonsRESUMO
An instrumental and experimental setup for soft x-ray spectroscopy meeting the requirements of a closed source for radioactivity is described. The system consists of a vacuum sealed cell containing the sample, mounted on a tubing system to ensure compatibility with most standard manipulators. The soft x rays penetrate a thin x-ray window separating the interior of the cell from the vacuum in the experimental chamber. Our first results for single crystal PuO(2) confirm the feasibility of experiments using the setup. The results are consistent with results of first principles calculations and previously recorded spectra obtained using a standard open source setup. The results show that the closed source experimental system can be used to collect valuable experimental data from radioactive materials.
RESUMO
Regulation of mitochondrial respiration both by endogenous and exogenous ADP in the cells in situ was studied in isolated and permeabilized cardiomyocytes, permeabilized cardiac fibers and 'ghost' fibers (all with a diameter of 10-20 micro m) at different (0-3 micro moll(-1)) free Ca(2+) concentrations in the medium. In all these preparations, the apparent K(m) of mitochondrial respiration for exogenous ADP at free Ca(2+) concentrations of 0-0.1 micro moll(-1) was very high, in the range of 250-350 micro moll(-1), in contrast to isolated mitochondria in vitro (apparent K(m) for ADP is approximately 20 micro moll(-1)). An increase in the free Ca(2+) concentration (up to 3 micro moll(-1), which is within physiological range), resulted in a very significant decrease of the apparent K(m) value to 20-30 micro moll(-1), a decrease of V(max) of respiration in permeabilized intact fibers and a strong contraction of sarcomeres. In ghost cardiac fibers, from which myosin was extracted but mitochondria were intact, neither the high apparent K(m) for ADP (300-350 micro moll(-1)) nor V(max) of respiration changed in the range of free Ca(2+) concentration studied, and no sarcomere contraction was observed. The exogenous-ADP-trapping system (pyruvate kinase + phosphoenolpyruvate) inhibited endogenous-ADP-supported respiration in permeabilized cells by no more than 40%, and this inhibition was reversed by creatine due to activation of mitochondrial creatine kinase. These results are taken to show strong structural associations (functional complexes) among mitochondria, sarcomeres and sarcoplasmic reticulum. Inside these complexes, mitochondrial functional state is controlled by channeling of ADP, mostly via energy- and phosphoryl-transfer networks, and apparently depends on the state of sarcomere structures.
Assuntos
Mitocôndrias Cardíacas/metabolismo , Células Musculares/metabolismo , Miofibrilas/metabolismo , Retículo Sarcoplasmático/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Respiração Celular/fisiologia , Cinética , Microscopia de Fluorescência , Miócitos Cardíacos/metabolismo , Ratos , Ratos Wistar , Sarcômeros/metabolismoRESUMO
In saponin-skinned muscle fibers from adult rat heart and m. soleus the apparent affinity of the mitochondrial oxidative phosphorylation system for ADP (Km = 200-400 microM) is much lower than in isolated mitochondria (Km = 10-20 microM). This suggests a limited permeability of the outer mitochondrial membrane (OMM) to adenine nucleotides in slow-twitch muscle cells. We have studied the postnatal changes in the affinity of mitochondrial respiration for ADP, in relation to morphological alterations and expression of mitochondrial creatine kinase (mi-CK) in rat heart in vivo. Analysis of respiration of skinned fibers revealed a gradual decrease in the apparent affinity of mitochondria to ADP throughout 6 weeks post partum that indicates the development of mechanism which increasingly limits the access of ADP to mitochondria. The expression of mi-CK started between the 1st and 2nd weeks and reached the adult levels after 6 weeks. This process was associated with increases in creatine-activated respiration and affinity of oxidative phosphorylation to ADP thus reflecting the progressive coupling of mi-CK to adenine nucleotide translocase. Laser confocal microscopy revealed significant changes in rearrangement of mitochondria in cardiac cells: while the mitochondria of variable shape and size appeared to be random-clustered in the cardiomyocytes of 1 day old rat, they formed a fine network between the myofibrils by the age of 3 weeks. These results allow to conclude that in early period of development, i.e. within 2-3 weeks, the diffusion of ADP to mitochondria becomes progressively restricted, that appears to be related to significant structural rearrangements such as formation of the mitochondrial network. Later (after 3 weeks) the control shifts to mi-CK, which by coupling to adenine nucleotide translocase, allows to maximally activate the processes of oxidative phosphorylation despite limited access of ADP through the OMM.
Assuntos
Difosfato de Adenosina/metabolismo , Creatina Quinase/metabolismo , Creatina/metabolismo , Coração/crescimento & desenvolvimento , Mitocôndrias Cardíacas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miocárdio/metabolismo , Fosforilação Oxidativa , Animais , Peso Corporal , Respiração Celular , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes , Coração/efeitos dos fármacos , Cinética , Microscopia Confocal , Mitocôndrias Musculares/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/citologia , Tamanho do Órgão , Ratos , Ratos Wistar , Tripsina/farmacologiaRESUMO
The kinetics of in vivo regulation of mitochondrial respiration by ADP was studied in rat heart, slow-twitch skeletal muscle (soleus) and fast-twitch skeletal muscle (gastrocnemius, plantaris, quadriceps and tibialis anterior) by means of saponin-skinned fibres. Mitochondrial respiratory parameters were determined in the absence and presence of creatine (20 mM), and the effect of proteolytic enzymes (trypsin, chymotrypsin or elastase) on these parameters was investigated in detail. The results of these experiments confirm the observation of Veksler et al. [Veksler, V.I., Kuznetsov, A. V., Anflous, K., Mateo, P., van Deursen, J., Wieringa, B. & Ventura-Clapier, R. (1995) J. Biol. Chem. 270, 19921-19929], who studied muscle fibres from normal and transgenic mice, that the kinetics of respiration regulation in muscle cells is tissue specific. We found that in rat cardiac and soleus muscle fibres the apparent K(m) for respiration regulation was 300-400 microM and decreased to 50-80 microM in the presence of creatine. In contrast, in skinned fibres from gastrocnemius, plantaris, tibialis anterior and quadriceps muscles, this value was initially very low, 10-20 microM, i.e. the same as that is in isolated muscle mitochondria, and the effect of creatine was not observable under these experimental conditions. Treatment of the fibres with trypsin, chymotrypsin or elastase (0.125 micrograms/ml) for 15 min decreased the apparent K(m) for ADP in cardiac and soleus muscle fibres to 40-98 microM without significant alteration of Vmax or the intactness of outer mitochondrial membrane, as assessed by the cytochrome c test. In fibres from gastrocnemius, trypsin increased the apparent K(m) for ADP transiently. The effects of trypsin and chymotrypsin were studied in detail and found to be concentration dependent and time dependent. The effects were characterised by saturation phenomenon with respect to the proteolytic enzyme concentration, saturation being observed above 1 microM enzyme. These results are taken to show that in cardiac and slow-twitch skeletal muscle, the permeability of the outer mitochondrial membrane to adenine nucleotides is low and controlled by a cytoplasmic protein that is sensitive to trypsin and chymotrypsin. This protein may participate in feedback signal transduction by a mechanism of vectorial-ligand conduction. This protein factor is not expressed in fast-twitch skeletal muscle, in which cellular mechanism of regulation of respiration is probably very different from that of slow-twitch muscles.
Assuntos
Difosfato de Adenosina/farmacologia , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Animais , Creatina/farmacologia , Coração/efeitos dos fármacos , Cinética , Mitocôndrias/metabolismo , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Fibras Musculares de Contração Lenta/ultraestrutura , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Ratos , Ratos Wistar , Serina Endopeptidases/farmacologia , Especificidade da Espécie , Distribuição TecidualRESUMO
The kinetics of regulation of mitochondrial respiration by endogenous and exogenous ADP in muscle cells in situ was studied in skinned cardiac and skeletal muscle fibres. Endogenous ADP production was initiated by addition of MgATP; under these conditions the respiration rate and ADP concentration in the medium were dependent on the calcium concentration, and 70-80% of maximal rate of respiration was achieved at ADP concentration below 20 microM in the medium. In contrast, when exogenous ADP was added, maximal respiration rate was observed only at millimolar concentrations. An exogenous ADP-consuming system consisting of pyruvate kinase (PK; 20-40 units/ml) and phosphoenolpyruvate (PEP; 5 mM), totally suppressed respiration activated by exogenous ADP, but the respiration maintained by endogenous ADP was not suppressed by more than 20-40%. Creatine (20 mM) further activated respiration in the presence of ATP and PK+PEP. Short treatment with trypsin (50-500 nM for 5 min) decreased the apparent K(m) for exogenous ADP from 300-350 microM to 50-60 microM, increased inhibition of respiration by PK+PEP system up to 70-80%, with no changes in MgATPase activity and maximal respiration rates. Electron-microscopic observations showed detachment of mitochondria and disordering of the regular structure of the sarcomere after trypsin treatment. Two-dimensional electrophoresis revealed a group of at least seven low-molecular-mass proteins in cardiac skinned fibres which were very sensitive to trypsin and not present in glycolytic fibres, which have low apparent K(m) for exogenous ADP. It is concluded that, in oxidative muscle cells, mitochondria are incorporated into functional complexes ('intracellular energetic units') with adjacent ADP-producing systems in myofibrils and in sarcoplasmic reticulum, probably due to specific interaction with cytoskeletal elements responsible for mitochondrial distribution in the cell. It is suggested that these complexes represent the basic pattern of organization of muscle-cell energy metabolism.