RESUMO
Oxidation-reduction changes of cytochrome P-450 and oxygen consumption were measured in isolated perfused livers from normal and phenobarbital-treated rats. Phenobarbital treatment markedly increased the aminopyrine-induced reduction of cytochrome P-450, but ethanol did not cause any redox changes of this cytochrome. It was concluded that the microsomal ethanol-oxidizing system has an insignificant role in the metabolism of ethanol in intact liver.
Assuntos
Citocromos/metabolismo , Etanol/metabolismo , Microssomos Hepáticos/metabolismo , Aminopirina/farmacologia , Animais , Monóxido de Carbono/farmacologia , Etanol/farmacologia , Técnicas In Vitro , Masculino , Oxirredução , Consumo de Oxigênio , Perfusão , Fenobarbital/administração & dosagem , Fenobarbital/farmacologia , Pigmentos Biológicos/metabolismo , RatosRESUMO
The heart muscle has proved to be a practical model for studying respiratory control in intact tissues. It also demonstrates that control at the level of the respiratory chain is augmented by metabolic control at the substrate level as exemplified by the very narrow range of changes in the redox state of the mitochondrial NADH/NAD couple even during extensive changes in ATP and oxygen consumption. The behaviour of mitochondria when isolated can largely be duplicated in the intact myocardium. Moreover, the high intracellular concentrations of enzymes, coenzymes and adenine nucleotides create conditions of high reaction rates, enabling the formation of a near equilibrium network of certain main pathways. This equilibrium network in connection with metabolic regulation of the hydrogen pressure upon the matrix NADH/NAD pool is a prerequisite for the regulation of cellular respiration at a high efficiency of energy transfer. Experimentation on the intact myocardium also seems to be capable of resolving some of the uncertainties about prevailing mechanisms for the regulation of cellular respiration.
Assuntos
Mitocôndrias Cardíacas/metabolismo , Consumo de Oxigênio , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Creatina Quinase/metabolismo , Eletroquímica , Metabolismo Energético , Hidrogênio/metabolismo , Técnicas In Vitro , Mitocôndrias Cardíacas/enzimologia , Modelos Biológicos , Miocárdio/citologia , Miocárdio/metabolismo , OxirreduçãoRESUMO
The sensitivity of the H+/2e- ratio of the redox-driven proton pumping by the NADH: ubiquinone reductase (complex I) of the submitochondrial particles to dicyclohexylcarbodiimide (DCCD) was studied by a thermodynamic approach, measuring the membrane potential and delta pH across the membrane and the redox potential difference across the complex I span of the respiratory chain. The delta Gr/delta muH+ ratio did not decrease upon additions of 50 or 100 nmol of DCCD per mg protein in the presence of oligomycin although the H+/2e- ratio has been demonstrated to decrease upon DCCD addition in kinetic experiments with mitochondria. Complex I then becomes reminiscent of the cytochrome bc1 complex, which shows DCCD sensitivity of the kinetically but not thermodynamically determined H+/2e- ratio.
Assuntos
Carbodi-Imidas/farmacologia , Dicicloexilcarbodi-Imida/farmacologia , Mitocôndrias Cardíacas/enzimologia , Quinona Redutases/antagonistas & inibidores , Partículas Submitocôndricas/enzimologia , Animais , Bovinos , Transporte de Elétrons/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , Potenciais da Membrana , Mitocôndrias Cardíacas/ultraestrutura , NAD(P)H Desidrogenase (Quinona) , Oligomicinas/farmacologia , Oxirredução , Prótons , Quinona Redutases/metabolismo , TermodinâmicaRESUMO
The effects of KCl-induced cardiac arrest on the redox state of the fluorescent flavoproteins and nicotinamide nucleotides and on that of cytochromes c and a were studied by surface fluorometric and reflectance spectrophotometric methods. These changes were compared with measurements of the concentrations of the adenylate system, creatine phosphate, some intermediates of the tricarboxylic acid cycle and reactants of the glutamate dehydrogenase system. KCl-induced cardiac arrest caused reduction of the fluorescent flavoproteins and nicotinamide nucleotides, oxidation of cytochromes c and a, inhibition of oxygen consumption and an increase in the ATP/(ADP X Pi) ratio. The increase in the latter was due mainly to a decrease in the concentration of Pi and an equivalent increase in creatine phosphate. The cytochromes c and a were maintained at equal redox potential and changed in parallel. When the redox state of the mitochondrial NAD couple was calculated from the glutamate dehydrogenase equilibrium, the free energy change (deltaG) corresponding to the potential difference between the NAD couple and cytochrome c was 115.8 kj/mol in the beating heart and 122.2 kj/mol in the arrested heart. The deltaG values of ATP hydrolysis calculated from the concentrations of ATP, Pi and ADP, corrected for bound ADP, were 111.1 kj/2 mol and 115.4 kj/2 mol in the beating and arrested heart respectively. The accumulation of citrate and the direction of the redox changes in the respiratory carriers indicate that the tricarboxylic acid cycle flux is controlled by the respiratory chain. The data also show a near equilibrium between the electron carriers and the adenylate system and suggest that the equilibrium hypothesis of mitochondrial respiratory control is applicable to intact myocardial tissue.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Mitocôndrias Musculares/metabolismo , Miocárdio/metabolismo , Consumo de Oxigênio , Animais , Calorimetria , Feminino , Parada Cardíaca/metabolismo , NAD/metabolismo , Oxirredução , Oxigênio/sangue , Pressão Parcial , Perfusão , Fosfatos/metabolismo , Ratos , Espectrometria de Fluorescência , TermodinâmicaRESUMO
1. The regulation of glycolysis and pyruvate oxidation under varying conditions of ATP and oxygen consumption was studied in isolated perfused rat hearts. Potassium-induced arrest was employed to inhibit the ATP consumption of the heart. 2. Under the experimental conditions, the beating heart used solely glucose as the oxidisable substrate. The glycolytic flux through the aldolase step decreased in pace with the decreasing oxygen consumption during the potassium-induced arrest of the heart. The decrease in glucose oxidation was larger than the inhibition of the oxygen consumption, suggesting that the arrested heart switches to fatty acid oxidation. The time course and percentage changes of the inhibition of pyruvate oxidation and the decrease in the amount of the active form of pyruvate dehydrogenase suggest that the amount of active pyruvate dehydrogenase is the main regulator of pyruvate oxidation in the perfused heart. 3. To test the relative significance of the possible mechanisms regulating covalent interconversions of pyruvate dehydrogenase, the following parameters were measured in response to the potassium-induced cardiac arrest: concentrations of pyruvate, acetyl-CoA, CoA-SH, citrate, alpha-oxoglutarate, ATP, ADP, AMP, creatine, creatine phosphate and inorganic phosphate and the mitochondrial NADH/NAD+ ratio. In cardiac tissue the adenylate system is not a good indicator of the energy state of the mitochondrion, even when the concentrations of AMP and free cytosolic ADP are calculated from the adenylate kinase and creatine kinase equilibria. Only creatine phosphate and inorganic phosphate undergo significant changes, but evidence of the participation of the latter compounds in the regulation of the pyruvate dehydrogenase interconversions is lacking. The potassium-induced arrest of the heart resulted in a decrease in pyruvate, a slight increase in acetyl-CoA, a large increase in the concentration of citrate and an increase in the mitochondrial NADH/NAD+. The results can be interpreted as showing that in the heart, the pyruvate dehydrogenase interconversions are mainly regulated by the pyruvate concentration and the mitochondrial redox state. Concentrations of all the regulators tested shifted to directions which one would expect to result in a decrease in the amount of active pyruvate dehydrogenase, but the changes were quite small. Therefore, the energy-linked regulation of pyruvate dehydrogenase in intact tissue is possibly mediated by the equilibrium relations between the cellular redox state and the phosphorylation potential recently confirmed in cardiac tissue.
Assuntos
Glicólise , Miocárdio/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Animais , Coenzima A/metabolismo , Feminino , Glucose/metabolismo , Cinética , Lactatos/metabolismo , Miocárdio/enzimologia , NAD/metabolismo , Oxirredução , Consumo de Oxigênio , Perfusão , Piruvatos/metabolismo , RatosRESUMO
The MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is most commonly caused by the 3243A-->G mutation in mitochondrial DNA, resulting in impaired mitochondrial protein synthesis and decreased activities of the respiratory chain complexes. These defects may cause a reduced capacity for ATP synthesis and an increased rate of production of reactive oxygen species. Myoblasts cultured from controls and patients carrying the 3243A-->G mutation were used to measure ATP, ADP, catalase and superoxide dismutase, which was also measured from blood samples. ATP and ADP concentrations were decreased in myoblasts with the 3243A-->G mutation, but the ATP/ADP ratio remained constant, suggesting a decrease in the adenylate pool. The superoxide dismutase and catalase activities were higher than in control cells, and superoxide dismutase activity was slightly, but not significantly higher in the blood of patients with the mutation than in controls. We conclude that impairment of mitochondrial ATP production in myoblasts carrying the 3243A-->G mutation results in adenylate catabolism, causing a decrease in the total adenylate pool. The increase in superoxide dismutase and catalase activities could be an adaptive response to increased production of reactive oxygen species due to dysfunction of the mitochondrial respiratory chain.
Assuntos
Trifosfato de Adenosina/metabolismo , Antioxidantes/metabolismo , DNA Mitocondrial/genética , Síndrome MELAS/enzimologia , Músculo Esquelético/enzimologia , Difosfato de Adenosina/metabolismo , Adulto , Catalase/metabolismo , Células Cultivadas , Feminino , Humanos , Síndrome MELAS/sangue , Síndrome MELAS/genética , Masculino , Pessoa de Meia-Idade , Fosforilação Oxidativa , Mutação Puntual , Superóxido Dismutase/sangue , Superóxido Dismutase/metabolismoRESUMO
The redox-mediated regulation of palmitoylcarnitine oxidation was studied in isolated rat liver mitochondria in which the mitochondrial free NADH/NAD+ ratio was controlled by graded concentrations of acetoacetate and ketomalonate in a rotenone and malonate-inhibited system in the presence of ADP. The NADH/NAD+ ratio was buffered kinetically by adjusting the concentrations of the hydrogen acceptor substances and determined by calibrated NAD(P)H fluorometry of the mitochondrial suspension. A two-fold variation in the beta-oxidation rate and a five-fold variation in the free NADH/NAD+ ratio was obtained in the presence of rotenone. A non-linear negative correlation was found between the acetyl-CoA concentration and the beta-oxidation rate and a negative correlation between the long-chain acyl-CoA concentration and the beta-oxidation rate. The data indicate that the redox state is a partial controller of the beta-oxidation rate in liver mitochondria. The contribution of acetyl-CoA, a putative regulator of beta-oxidation at the acyl-CoA thiolase step is small under the conditions used.
Assuntos
Carnitina/análogos & derivados , Mitocôndrias Hepáticas/metabolismo , NAD/metabolismo , Palmitoilcarnitina/metabolismo , Acetoacetatos/metabolismo , Acetilcoenzima A/metabolismo , Acil Coenzima A/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Grupo dos Citocromos c/metabolismo , Fluorometria , Masculino , Malonatos/metabolismo , Malonatos/farmacologia , Modelos Biológicos , NADP/metabolismo , Oxirredução , Ratos , Ratos Endogâmicos , Rotenona/metabolismoRESUMO
In view of its vasodilatory effect on the coronary circulation (probably mediated by adenosine) and its metabolic compartmentalization (intramitochondrial activation to form acetyl-CoA), the metabolic effects of acetate were studied in isolated rat heart mitochondria. Acetate caused conversion of adenylates to AMP and the formation of adenosine. Adenylate efflux was inhibited by carboxyatractyloside but not by N-ethylmaleimide. The intramitochondrial accumulation of AMP was enhanced by carboxyatractyloside during acetate metabolism and the formation of extramitochondrial adenosine inhibited. A carboxyatractyloside-sensitive unidirectional AMP influx with a Km of 50 microM and Vmax of 11 nmol/min per mg mitochondrial protein was also observed. The mitochondrial adenosine content was high and constant during the experiments. The steep apparent concentration gradient of adenosine indicates that most of the mitochondrial adenosine is tightly bound to protein. Adenosine formation was proportional to the extramitochondrial AMP concentration, showing that the 5'-nucleotidase activity of cardiac mitochondrial preparations is extramitochondrial in origin. The data suggest that the mitochondrial ATP/ADP carrier is capable of transporting AMP and that intramitochondrial AMP is recycled during acetate metabolism in the myocardium partially by means of the ATP/ADP translocator, leading to an increase in extramitochondrial AMP and adenosine formation.
Assuntos
Acetatos/metabolismo , Nucleotídeos de Adenina/metabolismo , Adenosina/biossíntese , Mitocôndrias Cardíacas/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Nucleotidiltransferases/metabolismo , 5'-Nucleotidase , Monofosfato de Adenosina/metabolismo , Animais , Técnicas In Vitro , Nucleotidases/metabolismo , Fosfatos/metabolismo , Piruvatos/metabolismo , RatosRESUMO
The possibility of a mutual regulation between carbohydrate and fatty acid oxidation was studied in isolated perfused rat hearts. Infusions of pyruvate and/or dichloroacetate were employed to convert fully the pyruvate dehydrogenase complex into its active form during [1-14C]octanoate oxidation, the rate of which was measured by the production of 14CO2. It was found that 5 mM dichloroacetate suppressed the oxidation of 0.1 mM octanoate by 58%, but 10 mM external pyruvate was without effect. Dichloroacetate reduced the tissue malate concentration by 41% and the citrate concentration by 76% at a 0.1 mM octanoate concentration, but had no effect on the metabolite concentrations or fatty acid oxidation rate in perfusions with 1 mM octanoate. Metabolite depletion was probably partly due to inhibition of the carboxylation of pyruvate, as verified by determination of the metabolite labelling kinetics from [1-14C]pyruvate. It is, therefore, possible that the dichloroacetate-induced inhibition of octanoate oxidation could also be partly due to inhibition of tricarboxylic acid cycle secondary to metabolite depletion. Since both dichloroacetate and pyruvate converted pyruvate dehydrogenase to its active form, but only dichloroacetate inhibited fatty acid oxidation, the latter effect could not be due to oxidation of a competing substrate, but instead may result from an inhibition of fatty acid uptake, activation or transport, as also indicated by the observed decrease in the acid-soluble acyl-CoA concentration. This interpretation is also supported by the mitochondrial redox effects of dichloroacetate observed during octanoate oxidation in the perfused heart.
Assuntos
Acetatos/farmacologia , Ácido Dicloroacético/farmacologia , Ácidos Graxos/metabolismo , Miocárdio/metabolismo , Piruvatos/farmacologia , Animais , Caprilatos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Masculino , Oxirredução/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The distribution of glutamate and aspartate and the mitochondrial membrane potential (delta psi) were studied in isolated rat heart mitochondria and in the intact perfused rat heart. The diffusion potential imposed by the glutamate-aspartate exchange through mediation of the electrogenic glutamate-aspartate translocator attained a value close to the mitochondrial delta psi measured from the distribution of triphenylmethylphosphonium ion (TPMP+) both in isolated mitochondria and in intact myocardium. Distributions of the delta psi probe and metabolites were determined by subcellular fractionation of the heart muscle in a non-aqueous medium. The results indicate that the glutamate-aspartate translocator is in near equilibrium in the myocardium. The diffusion potential of the glutamate-aspartate exchange, and the mitochondrial/cytosolic difference in the redox potentials of the free NAD+/NADH pools are equal allowing for experimental error. These data obtained from intact tissue can therefore be interpreted as supporting the notion of the transmembrane uphill transport of reducing equivalent from the cytosolic free NAD+/NADH pool being driven by the malate-aspartate cycle energized by the mitochondrial delta psi.
Assuntos
Mitocôndrias Cardíacas/fisiologia , Sistema X-AG de Transporte de Aminoácidos , Animais , Ácido Aspártico/metabolismo , Transporte Biológico Ativo , Glutamatos/metabolismo , Membranas Intracelulares/fisiologia , Masculino , Potenciais da Membrana , Miocárdio/metabolismo , NAD/fisiologia , Oxirredução , RatosRESUMO
Subcellular fractionation of tissue in nonaqueous media was employed to study metabolite compartmentation in isolated perfused rat hearts. The mitochondrial and cytosolic concentrations of citrate and 2-oxoglutarate, total concentrations of the glycolytic intermediates and rate of glycolysis were measured in connection with changes in the rate of cellular respiration upon modulation of the ATP consumption by changes of the mechanical work load of the heart. The concentrations of citrate and 2-oxoglutarate in the mitochondria were 16- and 14-fold, respectively, greater than those in the cytosol of beating hearts. The cytosolic citrate concentration was low compared with concentrations which have been employed in demonstrations of the citrate inhibition of glycolysis. In spite of the low activities reported for the tricarboxylate carrier in heart mitochondria, the cytosolic citrate concentration reacted to perturbations of the mitochondrial citrate concentration, and inhibition of glycolysis at the phosphofructokinase step could be observed concomitantly with an increase in the cytosolic citrate concentration. The delta pH across the inner mitochondrial membrane calculated from the 2-oxoglutarate concentration gradient and the mitochondrial membrane potential calculated from the adenylate distribution gave an electrochemical potential difference of protons compatible with chemiosmotic coupling in the intact myocardium.
Assuntos
Glicólise , Membranas Intracelulares/fisiologia , Mitocôndrias Cardíacas/fisiologia , Animais , Transporte Biológico Ativo , Citratos/metabolismo , Ácido Cítrico , Feminino , Concentração de Íons de Hidrogênio , Cinética , Potenciais da Membrana , Consumo de Oxigênio , Ratos , Ratos EndogâmicosRESUMO
Tricarboxylic acid cycle pool size is determined by anaplerosis and metabolite disposal. The regulation of the latter during propionate metabolism was studied in isolated perfused rat hearts in the light of the characteristics of NADP-linked malic enzyme, which is inhibited by acetyl-CoA. The acetyl-CoA concentration was varied by infusions of acetate and octanoate, and the rate of metabolite disposal was calculated from a metabolic balance sheet compiled from the relevant metabolic fluxes. Propionate addition increased the tricarboxylic acid cycle pool size 4-fold and co-infusion of acetate or octanoate did not change it further. Propionate caused a decrease in the CoA-SH concentration and a 10-fold increase in the propionyl-CoA concentration. A paradoxical increase in the CoA-SH concentration was observed upon co-infusion of acetate in the presence of propionate, an effect probably caused by competitive inhibition of propionate activation. A more pronounced decline in the propionyl-CoA concentration was observed upon the co-infusion of octanoate. In a metabolic steady state, acetate and octanoate reduced propionate disposal only slightly, but did not increase the tricarboxylic acid cycle pool size. The results are in accord with the notion that the tricarboxylic acid pool size is mainly regulated by the anaplerotic mechanisms.
Assuntos
Acetatos/farmacologia , Caprilatos/farmacologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Miocárdio/metabolismo , Propionatos/metabolismo , Ácido Acético , Acetilcoenzima A/metabolismo , Acil Coenzima A/metabolismo , Animais , Coenzima A/metabolismo , Feminino , Glicogênio/metabolismo , Glicólise , Técnicas In Vitro , Malatos/metabolismo , Oxirredução , Consumo de Oxigênio , Perfusão , Complexo Piruvato Desidrogenase/metabolismo , Ratos , Ratos Endogâmicos , Succinatos/metabolismo , Ácido SuccínicoRESUMO
The controversial subject of mitochondrial 5'-nucleotidase in the liver was studied employing density gradient fractionation combined with a method for analyzing the distribution profiles of marker enzymes based on multiple regression analysis. Triton WR-1339 was used to improve the separation of mitochondria from lysosomes by the gradient centrifugation technique. Adenosine production was examined further using acetate to increase intramitochondrial AMP, and thus adenosine production, in incubations with gradient centrifugation-purified mitochondria. Distribution analysis of the crude homogenate showed that 5'-nucleotidase activity exists in the mitochondrial fraction. To increase the resolution of this approach with respect to mitochondria, a crude mitochondrial fraction was also studied. In this case the relative mitochondrial activity decreased but 5'-nucleotidase activity was still clearly detectable. The mitochondrial 5'-nucleotidase exhibited a Km of 94 microM and a Vmax of 31 nmol/min per mg protein for AMP. The kinetic data for the Mg2+, ATP, ADP and AOPCP sensitivity of the enzyme showed that it differs from the plasma membrane, lysosome and cytosol 5'-nucleotidases. AOPCP was only a moderate inhibitor, and ATP was a more potent inhibitor than ADP at a 1 mM concentration. The enzyme also showed a requirement of Mg2+. Acetate caused the conversion of intramitochondrial adenylates to AMP and the formation of adenosine. Adenosine concentration increased in the extramitochondrial space in a time-dependent manner, but only trace amounts of nucleotides were detected. The data show that 5'-nucleotidase activity producing adenosine exists in rat liver mitochondria and a concentration-dependent adenosine output from mitochondria by diffusion or facilitated diffusion is also suggested.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina/biossíntese , Mitocôndrias Hepáticas/metabolismo , Acetatos/farmacologia , Animais , Cinética , Masculino , Piruvatos/farmacologia , Ácido Pirúvico , Ratos , Ratos Endogâmicos , Análise de Regressão , Frações Subcelulares/enzimologiaRESUMO
OBJECTIVES: The aim of this study was to assess the occurrence of the two most commonly encountered mitochondrial DNA (mtDNA) deletions in the hearts of patients with idiopathic dilated cardiomyopathy. BACKGROUND: The mutation frequency of mtDNA is high, and sporadic cases of cardiomyopathies associated with mtDNA deletions have been described. Reports of increases in mtDNA deletions with advancing age also exist. METHODS: We studied 15 consecutive patients with typical signs of idiopathic dilated cardiomyopathy, without a family history, together with 16 control hearts obtained at autopsy from patients who died of noncardiac causes. The patients underwent both right and left heart catheterization, during which endomyocardial biopsy samples were taken. The mtDNA in these samples and in the control hearts was analyzed by the polymerase chain reaction technique for the occurrence and proportion of 5- and 7.4-kilobase (kb) deletions (Cambridge sequence map positions from nucleotides 8469 to 13447 and 8637 to 16084, respectively). RESULTS: The 5-kb mtDNA deletion was observed in the hearts of all of the patients with idiopathic dilated cardiomyopathy, accounting for 0.32 +/- 0.05% (mean +/- SEM) of the total mtDNA. The 7.4-kb deletion was found in 7 of the 15 patients with idiopathic dilated cardiomyopathy and comprised 0.28 +/- 0.08% of the total. The 5- and 7.4-kb deletions were detected in 12 and 9 control hearts, respectively, quantitatively similar to the patients with idiopathic dilated cardiomyopathy. A sigmoidal age dependency of the mtDNA deletions was found both in the patients with cardiomyopathy and in the control hearts, but after elimination of the confounding age variable, there was no difference between these groups. CONCLUSIONS: Because of the similarity of the age-dependent increase in the frequency of mtDNA deletions in cardiomyopathic and control hearts, the deletions have no causal relation with idiopathic dilated cardiomyopathy. The present results confirm the notion of an increase in mtDNA deletions with advancing age and show that endomyocardial tissue sampling is a feasible method for detecting mtDNA defects in affected hearts.
Assuntos
Cardiomiopatia Dilatada/genética , DNA Mitocondrial/genética , Deleção de Genes , Adulto , Fatores Etários , Cardiomiopatia Dilatada/patologia , Estudos de Casos e Controles , Mapeamento Cromossômico , Endocárdio/ultraestrutura , Feminino , Humanos , Modelos Logísticos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
Reflectance spectrophotometry was assessed as a means of detecting metabolic oxidation-reduction changes in cytochromes and the oxygenation level of myoglobin in isolated perfused rat hearts. The movement artefact due to the heartbeat was largely eliminated by appropriate design of the optical geometry. A considerable myocardial oxygen gradient was detected using myoglobin deoxygenation as an indicator. The oxygenation level of myoglobin oscillated in pace with the contraction/relaxation cycle of the heart, deoxygenation occurring during the systole. The inconsistency in the previous reports on the myoglobin oxygenation and the problems of spectrophotometry of the beating heart are discussed.
Assuntos
Citocromos/metabolismo , Miocárdio/metabolismo , Mioglobina/metabolismo , Espectrofotometria/métodos , Animais , Pressão Sanguínea , Masculino , Contração Miocárdica , Oxirredução , Oxigênio/sangue , Consumo de Oxigênio , Perfusão , Ratos , Espectrofotometria/instrumentação , Função VentricularRESUMO
Using a new method of culturing neonatal rat heart muscle cells on collagen coated Sephadex microspheres the culture showed increasing biomass for 72 h, at which time 44% of the cells were myocytes. At 24 h after inoculation 63% of the cells were myocytes, and from 48 h onwards they were beating spontaneously. The cell yield was sufficient for metabolite determination by enzymatic cycling and fluorometric methods. The ease of manipulation of the cells on the microspheres gives this method considerable potential for studies of compartmentation, biosynthesis, and secretion in cardiac myocytes.
Assuntos
Miocárdio/ultraestrutura , Animais , Células Cultivadas , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Microesferas , Miocárdio/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Regulation of atrial natriuretic peptide (ANP) secretion from neonatal rat myocytes cultured on microcarriers was studied using endothelin-1 (ET-1) as a secretagogue. Myocytes were cultured for 3 days on microcarriers, packed in a chromatography column, and perifused with Krebs-Henseleit bicarbonate buffer. ANP secretion was measured by RIA, and the cytosolic free calcium concentration ([Ca2+]f) was measured continuously during secretion by the fluorescent calcium indicator fura-2. In perifused atrial and ventricular cells, basal values for [Ca2+]f were 146 and 167 nM, and immunoreactive ANP (IR-ANP) secretion rates were 61 and 65 pg/min.mg protein, respectively. ET-1 at concentrations of 1, 10, and 100 nM caused a concentration-dependent increases in [Ca2+]f and IR-ANP secretion in atrial myocytes. The maximal increases in [Ca2+]f and IR-ANP secretion were 30% and 100%, respectively. Diltiazem (1 microM), an inhibitor of voltage-sensitive Ca2+ channels, inhibited [Ca2+]f increments, but had no effect on ET-induced IR-ANP secretion. Staurosporine (10 nM), a protein kinase-C inhibitor, augmented [Ca2+]f changes, but inhibited the sustained phase of ET-induced IR-ANP secretion (P less than 0.05). Diltiazem abolished the stimulatory effect of staurosporine on [Ca2+]f and its inhibitory effect on IR-ANP secretion. ET-1 caused increases in [Ca2+]f and IR-ANP secretion in ventricular myocytes similar to those in atrial myocytes. Peptides corresponding in size to pro-ANP and ANP-(1-28) were detected in the original cell culture medium and perifusion effluent, and ET-1 did not change their concentration ratio in the eluate. Lactate dehydrogenase was not detected in the effluents before or during ET infusion, showing that the increase in IR-ANP secretion was not due to cell damage. This study shows that ET stimulates atrial and ventricular ANP secretion. The results also suggest that sustained ET-induced atrial ANP secretion is dependent on protein kinase-C, but does not require the influx of extracellular calcium.
Assuntos
Fator Natriurético Atrial/metabolismo , Cálcio/metabolismo , Endotelinas/farmacologia , Coração/fisiologia , Proteína Quinase C/metabolismo , Alcaloides/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Fator Natriurético Atrial/biossíntese , Células Cultivadas , Diltiazem/farmacologia , Coração/efeitos dos fármacos , Indometacina/farmacologia , Cinética , L-Lactato Desidrogenase/metabolismo , Miocárdio/metabolismo , Perfusão , Proteína Quinase C/antagonistas & inibidores , Radioimunoensaio , Ratos , EstaurosporinaRESUMO
Several point mutations in mitochondrial DNA (mtDNA) have been shown to cause sensorineural hearing impairment (SNHI), but the frequency of these mutations among patients is not known. We identified 117 patients with possible matrilineal SNHI from population-based registers and found the 3243A > G mutation to be present in 4.3% and 1555A > G in 2.6%, while 7445T > C, 7472insC and 8344A > G were absent. Patients with 3243A > G and 1555A > G were clinically distinct. The prevalence of 1555A > G in the general adult population was estimated to be at least 4.7/100,000, but these and previous data suggest that the figure may vary between populations. Screening for mtDNA mutations is worthwhile in connection with the diagnosis of SNHI.
Assuntos
DNA Mitocondrial/genética , Perda Auditiva Neurossensorial/genética , Estudos de Coortes , Saúde da Família , Feminino , Frequência do Gene , Haplótipos , Perda Auditiva Neurossensorial/patologia , Testes Auditivos , Humanos , Masculino , Linhagem , Mutação PuntualRESUMO
The metabolism of the double bonds at the delta 3 position in fatty acids was studied in rat liver. Infusion of delta 3-trans-dodecenoic acid into isolated perfused liver and subcellular fractionation studies showed the presence of both peroxisomal and mitochondrial delta 3,delta 2-enoyl-CoA isomerase activity (EC 5.3.3.8). These findings together with the previous demonstration of peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34) [(1981) J. Biol. Chem. 256, 8259-8262] and D-3-OH-acyl-CoA epimerase (EC 5.1.2.3) [(1985) FEBS Lett. 185, 129-134] activities show that peroxisomes possess all the auxiliary enzymes required for the beta-oxidation of unsaturated fatty acids.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono , Ácidos Graxos Insaturados/metabolismo , Isomerases/metabolismo , Fígado/enzimologia , Frações Subcelulares/enzimologia , Animais , Centrifugação , Dodecenoil-CoA Isomerase , Masculino , Microcorpos/enzimologia , Mitocôndrias Hepáticas/enzimologia , Oxirredução , Ratos , Ratos EndogâmicosRESUMO
The activities of four lysosomal and two nonlysosomal hydrolases were studied in skeletal muscle biopsy samples from patients with neuromuscular diseases and from controls. beta-Glucosaminidase activity was increased in polymyositis. beta-Glucuronidase and alkaline protease activities were elevated in muscular dystrophy in adults, whereas cathepsin D activity was increased in amyotrophic lateral sclerosis. There were significant correlations between the activities of lysosomal and nonlysosomal hydrolases. The activity of beta-glucuronidase, beta-glucosaminidase, alkaline protease, and dipeptidyl aminopeptidase IV showed a positive correlation with the severity of muscular atrophy. The activities of these hydrolases and the activity of dipeptidyl aminopeptidase I correlated positively with the activities of muscular galactosylhydroxylysyl glucosyltransferase and with the serum concentration of type III procollagen aminoterminal propeptide. The results suggest that in neuromuscular diseases the lysosomal and nonlysosomal pathways for muscle degradation are affected concomitantly with collagen biosynthesis.