RESUMEN

How the myocardium is able to permanently coordinate its intracellular fluxes of ATP synthesis, transfer and utilization is difficult to investigate in the whole organ due to the cellular complexity. The adult myocardium represents a paradigm of an energetically compartmented cell since 50% of total CK activity is bound in the vicinity of other enzymes (myofibrillar sarcolemmal and sarcoplasmic reticulum ATPases as well as mitochondrial adenine nucleotide translocator, ANT). Such vicinity of enzymes is well known in vitro as well as in preparations of skinned fibers to influence the kinetic properties of these enzymes and thus the functioning of the subcellular organelles. Intracellular compartmentation has often been neglected in the NMR analysis of CK kinetics in the whole organ. It is indeed a methodological challenge to reveal subcellular kinetics in a working organ by a global approach such as NMR. To get insight in the energy transfer pathway in the perfused rat heart, we developed a combined analysis of several protocols of magnetization transfer associated with biochemical data and quantitatively evaluated which scheme of energetic exchange best describes the NMR data. This allows to show the kinetic compartmentation of subcellular CKs and to quantify their fluxes. Interestingly, we could show that the energy transfer pathway shifts from the phosphocreatine shuttle in the oxygenated perfused heart to a direct ATP diffusion from mitochondria to cytosol under moderate inhibition of ATP synthesis. Furthermore using NMR measured fluxes and the known kinetic properties of the enzymes, it is possible to model the system, estimate local ADP concentrations and propose hypothesis for the versatility of energy transfer pathway. In the normoxic heart, a 3-fold ADP gradient was found between mitochondrial intermembrane space, cytosol and ADP in the vicinity of ATPases. The shift from PCr to ATP transport observed when ATP synthesis decreases might result from a balance in the activity of two populations of ANT, either coupled or uncoupled to CK. We believe this NMR approach could be a valuable tool to reinvestigate the control of respiration by ADP in the whole heart reconciling the biochemical knowledge of mitochondrial obtained in vitro or in skinned fibers with data on the whole heart as well as to identify the implication of bioenergetics in the pathological heart.

Asunto(s)

Adenosina Trifosfato/metabolismo , Creatina Quinasa/metabolismo , Miocardio/metabolismo , Transferencia de Energía , Espectroscopía de Resonancia Magnética , Orgánulos/enzimología , Orgánulos/metabolismo

RESUMEN

The exchange scheme of high energy phosphate transport in a whole heart relies on a system of CK functioning in different ways. This suggests that the CKs are able to act both like a shuttle and like a buffer for the energy transfer. The challenge is to understand how these two functions are balanced in the CK system. One key of this balance is the knowledge of the local concentrations of the ADP nucleotide. These concentrations cannot be directly measured, but they may be derived by computation. In the present report we introduce the known properties of the enzymes catalyzing the exchange of high energy phosphate into the model of flux pathways derived from NMR experiments to compute both the maximum activity of each enzyme and the local concentrations of all the substrates. We show that the ADP distribution must be heterogeneous for the system to work. Its concentration is 50% higher in the vicinity of ATPase sites and 50% lower in the intermembrane space of the mitochondria than in the cytosol. Another result of this analysis is that the apparent large unbalance of the CKmito pathway is imposed by the adenosine nucleotide transferase fluxes. This analysis proves that it is possible to deduce biochemistry the local concentrations of a substrate by combining data originating from NMR, and enzymology into a common model.

Asunto(s)

Adenosina Difosfato/metabolismo , Creatina Quinasa/metabolismo , Metabolismo Energético , Modelos Cardiovasculares , Miocardio/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Corazón/fisiología , Técnicas In Vitro , Matemática , Resonancia Magnética Nuclear Biomolecular , Perfusión , Ratas

RESUMEN

The identification of subcellular fluxes of exchange of ATP, phosphocreatine (PCr) and Pi between mitochondria, cytosol and ATPases and pathways of energy transfer in a whole organ is a challenge specially in the myocardium where 50% of creatine kinases (CK) are found in close vicinity of ATP producing (mito-CK) and utilizing (MM-bound CK) reactions. To dissect their contribution in cardiac energy transfer we recently developed a new experimental 31P NMR spectroscopy approach. This led to identify three kinetically different subcellular CKs and to evidence experimentally the CK shuttle in a rat heart perfused in isovolumy. Here we show that a decreased energy demand alters energetic pathways : two CKs (cytosolic and MM-bound) functioning at equilibrium and a non mitochondrial ATP<-->Pi exchange was sufficient to describe NMR data. Mito-CK fluxes was not detected anymore. This confirms the dependence of energy pathways upon cardiac activity. Indeed the subcellular localization and activity of CKs may have important bioenergetic consequences for the in vivo control of respiration at high work: free ADP estimated from global CK equilibrium might not always adequately reflect its concentration at the ANT.

Asunto(s)

Metabolismo Energético , Corazón/fisiología , Mitocondrias Cardíacas/metabolismo , Contracción Miocárdica/fisiología , Adenosina Trifosfato/metabolismo , Algoritmos , Animales , Creatina Quinasa/metabolismo , Técnicas In Vitro , Matemática , Resonancia Magnética Nuclear Biomolecular , Perfusión , Ratas

RESUMEN

A challenge in the understanding of creatine kinase (CK) fluxes reflected by NMR magnetization transfer in the perfused rat heart is the choice of a kinetic model of analysis. The complexity of the energetic pathways, due to the presence of adenosine triphosphate (ATP)-inorganic phosphate (Pi) exchange, of metabolite compartmentation and of subcellular localization of CK isozymes cannot be resolve from the sole information obtained from a single NMR protocol. To analyze multicompartment exchanges, we propose a new strategy based on the simultaneous analysis of four inversion transfer protocols. The time course of ATP and Phosphocreatine (PCr) magnetizations computed from the McConnell equations were adjusted to their experimental value for exchange networks of increasing complexity (up to six metabolite pools). Exchange schemes were selected by the quality of their fit and their consistency with data from other sources: the size of mitochondrial pools and the ATP synthesis flux. The consideration of ATP-Pi exchange and of ATP compartmentation were insufficient to describe the data. The most appropriate exchange scheme in our normoxic heart involved the discrimination of three specific CK activities (cytosolic, mitochondrial, and close to ATPases). At the present level of heart contractility, the energy is transferred from mitochondria to myofibrils mainly by PCr.

Asunto(s)

Adenosina Trifosfato/metabolismo , Creatina Quinasa/biosíntesis , Creatina Quinasa/química , Miocardio/enzimología , Animales , Distribución de Chi-Cuadrado , Transferencia de Energía , Cinética , Espectroscopía de Resonancia Magnética , Masculino , Modelos Biológicos , Modelos Químicos , Modelos Estadísticos , Perfusión , Ratas , Ratas Wistar , Programas Informáticos

RESUMEN

In the perfused rat heart NMR inversion transfer revealed the existence of a compartment of ATP not exchanging through creatine kinase (CK), as demonstrated by an apparent discrepancy between the forward (F(f)) and reverse (F(r)) CK flux if this compartment was neglected in the analysis [Joubert et al. (2000) Biophys. J. 79, 1-13]. To localize this compartment, CK fluxes were measured by inversion of PCr (inv-PCr) or gamma ATP (inv-ATP), and the distribution of metabolites between mitochondria and cytosol was studied by subcellular fractionation. Physiological conditions were designed to modify the concentration and distribution of CK metabolites (control, adenylate depletion, inhibition of respiration, KCl arrest). Depending on cardiac activity, mitochondrial ATP (mito-ATP) assessed by fractionation varied from 11% to 30% of total ATP. In addition, the apparent flux discrepancy increased together with mito-ATP (F(f)/F(r) ranged from 0.85 to 0.50 in inv-PCr and from 1.13 to 1.88 in inv-ATP). Under conditions masking the influence of the ATP-P(i) exchange on CK flux, the ATP compartment could be directly quantified by the apparent flux discrepancy; its size was similar to that of mito-ATP measured by fractionation. Thus NMR inversion technique is a potential tool to assess metabolite compartmentation in the whole organ.

Asunto(s)

Creatina Quinasa/metabolismo , Mitocondrias Cardíacas/enzimología , Miocardio/enzimología , Adenosina Trifosfato/metabolismo , Animales , Técnicas In Vitro , Líquido Intracelular/enzimología , Líquido Intracelular/metabolismo , Masculino , Mitocondrias Cardíacas/metabolismo , Modelos Biológicos , Contracción Miocárdica , Miocardio/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Ratas , Ratas Wistar , Partículas Submitocóndricas/enzimología , Partículas Submitocóndricas/metabolismo

RESUMEN

The interpretation of creatine kinase (CK) flux measured by (31)P NMR magnetization transfer in vivo is complex because of the presence of competing reactions, metabolite compartmentation, and CK isozyme localization. In the isovolumic perfused rat heart, we considered the influence of both ATP compartmentation and ATP-P(i) exchange on the forward (F(f): PCr --> ATP) and reverse (F(r)) CK fluxes derived from complete analysis of inversion transfer. Although F(f) should equal F(r) because of the steady state, in both protocols when PCr (inv-PCr) or ATP (inv-ATP) was inverted and the contribution of ATP-P(i) was masked by saturation of P(i) (sat-P(i)), F(f)/F(r) significantly differed from 1 (0.80 +/- 0.06 or 1.32 +/- 0.06, respectively, n = 5). These discrepancies could be explained by a compartment of ATP (f(ATP)) not involved in CK. Consistently, neglecting ATP compartmentation in the analysis of CK in vitro results in an underestimation of F(f)/F(r) for inv-PCr and its overestimation for inv-ATP. Both protocols gave access to f(ATP) if the system was adequately analyzed. The fraction of ATP not involved in CK reaction in a heart performing medium work amounts to 20-33% of cellular ATP. Finally, the data suggest that the effect of sat-P(i) might not result only from the masking of ATP-P(i) exchange.

Asunto(s)

Adenosina Trifosfato/metabolismo , Creatina Quinasa/metabolismo , Miocardio/enzimología , Adenosina Trifosfato/farmacología , Animales , Compartimento Celular/efectos de los fármacos , Compartimento Celular/fisiología , Intervalos de Confianza , Corazón/efectos de los fármacos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética/métodos , Masculino , Modelos Cardiovasculares , Miocardio/citología , Perfusión , Fosfatos/metabolismo , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Sensibilidad y Especificidad

RESUMEN

We have tested the hypothesis that decreased functioning of creatine kinase (CK) at sites of energy production and utilization may contribute to alterations in energy fluxes and calcium homeostasis in congestive heart failure (CHF). Heart failure was induced by aortic banding in 3-week-old rats. Myofilaments, sarcoplasmic reticulum (SR), mitochondrial functions, and CK compartmentation were studied in situ using selective membrane permeabilization of left ventricular fibers with detergents (saponin for mitochondria and SR and Triton X-100 for myofibrils). Seven months after surgery, animals were in CHF. A decrease in total CK activity could be accounted for by a 4-fold decrease in activity and content (Western blots) of mitochondrial CK and a 30% decrease in M isoform of CK (MM-CK) activity. In myofibrils, maximal force, crossbridge kinetics, and alpha-myosin heavy-chain expression decreased, whereas calcium sensitivity of tension development remained unaltered. Myofibrillar CK efficacy was unchanged. Calcium uptake capacities of SR were estimated from the surface of caffeine-induced tension transient (SCa) after loading with different substrates. In CHF, SCa decreased by 23%, and phosphocreatine was 2 times less efficient in enhancing calcium uptake. Oxidative capacities of the failing myocardium measured as oxygen consumption per gram of fiber dry weight decreased by 28%. Moreover, the control of respiration by creatine, ADP, and AMP was severely impaired. Our observations provide evidence that alterations in CK compartmentation may contribute to alterations of energy fluxes and calcium homeostasis in CHF.

Asunto(s)

Creatina Quinasa/metabolismo , Insuficiencia Cardíaca/enzimología , Miocardio/enzimología , Fracciones Subcelulares/enzimología , Animales , Corazón/fisiopatología , Insuficiencia Cardíaca/fisiopatología , Masculino , Mitocondrias Cardíacas/fisiología , Miofibrillas/fisiología , Ratas , Ratas Wistar , Retículo Sarcoplasmático/fisiología , Función Ventricular Izquierda/fisiología

RESUMEN

To study the relation among mitochondrial energy supply, cardiac performance, and energy transfer through creatine kinase (CK), two acute models of inhibition of ATP synthesis were compared in the isovolumic acetate-perfused rat heart. Similar impairments of mechanical performance (rate-pressure product, RPP) were achieved by various stepwise decreases in O(2) supply (PO(2) down to 20% of control) or by infusing CN (0.15-0.25 mM). The forward CK flux measured by saturation-transfer (31)P NMR spectroscopy was 6.1 +/- 0. 4 mM/s in control hearts. Only after severe hypoxia (PO(2) < 40% of control) did CK flux drop (to 1.9 +/- 0.2 mM/s at PO(2) = 25% of control) together with impaired systolic activity and a rise in end-diastolic pressure. In contrast, in mild hypoxia CK flux remained constant and similar to control (5.3 +/- 0.5 mM/s, not significant) despite a twofold reduction in systolic activity. Similarly in all CN groups, constant CK flux was maintained for a threefold reduction in RPP, showing the absence of a relation between cardiac performance and global NMR-measured CK flux during mild ATP synthesis inhibition.

Asunto(s)

Adenosina Trifosfato/biosíntesis , Creatina Quinasa/metabolismo , Corazón/fisiología , Miocardio/enzimología , Animales , Hipoxia/metabolismo , Cinética , Masculino , Oxígeno/metabolismo , ATPasas de Translocación de Protón/metabolismo , Ratas , Ratas Wistar

RESUMEN

We have investigated the effect of chronic exposure of rats to an hypoxic environment (10% O2; 3 weeks), on the first step of the intracellular energy transfer process in the myocardium, i.e. the transfer at mitochondrial level of high energy bonds from ATP to creatine. In the left ventricles from rats adapted to normobaric hypoxia, we observed, using the permeabilized fiber technique, that the stimulatory effect of creatine on the mitochondrial respiration in presence of a low ADP concentration (0.1 mM) was attenuated when compared to control. Furthermore, the creatine-induced decrease of the apparent K(m) for ADP of the mitochondrial respiration, which is observed in control, was significantly reduced. Both the basal and maximal respiratory rates of the fibers were unchanged by the hypoxic exposure of the rats. A significant decrease of the total creatine kinase activity from 755 to 630 IU/g wet weight (for control and hypoxic rats, respectively) was detected and was accompanied by a 25% decrease in mitochondrial isoform activity (mitoCK) and in the mitoCK/citrate synthase ratio. In the right ventricles, identical alterations in the effect of creatine on apparent K(m) for ADP were observed while we did not detect any changes in CK activity. The decrease in mitoCK activity and the fall in the reactivity of respiration to creatine could be interpreted as a mechanism for downregulating oxygen demand during chronic hypoxia. The consequences of such alterations on energy metabolism of cardiomyocytes under conditions of reduced oxygen supply are discussed.

Asunto(s)

Transferencia de Energía , Miocardio/metabolismo , Oxígeno/metabolismo , Adenosina Difosfato/metabolismo , Adenilato Quinasa/metabolismo , Animales , Creatina/metabolismo , Creatina Quinasa/metabolismo , Femenino , Tabiques Cardíacos/fisiología , Mitocondrias/enzimología , Tamaño de los Órganos , Fosfatos/metabolismo , Ratas , Ratas Wistar , Función Ventricular

RESUMEN

rpoS homologues were identified in several Erwinia species using Escherichia coli rpoS sequences as probes. The rpoS gene from Erwinia carotovora was cloned and the deduced amino acid sequence had 91% identity to E. coli RpoS. The latter sigma factor regulates the stationary phase inducible HPII catalase activity of E. coli. In an E. coli rpoS mutant, the E. carotovora rpoS gene was also able to regulate synthesis of this catalase. The presence of a similar catalase in E. carotovora suggests that the structural gene for this may be part of the rpoS 'regulon' in Erwinia also. This study also showed that there are several differences in the gene organization of the rpoS region of the E. coli and E. carotovora chromosomes.

Asunto(s)

Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Bacterianos , Pectobacterium carotovorum/genética , Factor sigma/genética , Secuencia de Aminoácidos , Secuencia de Bases , Catalasa/metabolismo , Clonación Molecular , Datos de Secuencia Molecular

RESUMEN

A new mathematical model, based on the observation of 13C-NMR spectra of two principal metabolites (glutamate and aspartate), was constructed to determine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate and glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considered to be interdependent. With [U-13C]Glc or [1,2-(13)C]acetate as a substrate, all glutamate and aspartate carbons can be labeled. The isotopic transformations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in two matrices. We showed how the flux constants of the citric acid cycle and the 13C-enrichment of acetyl-CoA can be deduced from 13C-NMR spectra of glutamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-13C]glucose in the absence of insulin, was investigated by 13C-NMR spectroscopy. Surprisingly, aspartate instead of glutamate was found to be the most abundantly-labeled metabolite, indicating that aspartate transaminase (which catalyses the reversible reaction: (glutamate + oxaloacetate <--> 2-oxoglutarate + aspartate) is highly active in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (G0) or aspartate (A0) were approximately the same for all hearts and remained constant during perfusion: G0 = (0.74 +/- 0.03) micromol/g; A0 = (1.49 +/- 0.05) micromol/g. The flux constants, i.e., the fraction of glutamate and aspartate in exchange with the citric acid cycle, were about 1.45 min(-1) and 0.72 min(-1), respectively; the flux of this cycle is about (1.07 +/- 0.02) micromol min(-1) g(-1). Excellent agreement between the computed and experimental data was obtained, showing that: i) in the absence of insulin, only 41% of acetyl-CoA is formed from glucose while the rest is derived from endogenous substrates; and ii) the exchange between aspartate and oxaloacetate or between glutamate and 2-oxoglutarate is fast in comparison with the biological transformation of intermediate compounds by the citric acid cycle.

Asunto(s)

Aspartato Aminotransferasas/metabolismo , Ciclo del Ácido Cítrico , Miocardio/enzimología , Acetilcoenzima A/metabolismo , Animales , Isótopos de Carbono , Glucosa/administración & dosificación , Espectroscopía de Resonancia Magnética , Masculino , Perfusión , Ratas , Ratas Wistar

RESUMEN

To study the dependence of the forward flux of creatine kinase (CK) on its substrates and products we designed an acute normoxic model of steady-state depletion of phosphocreatine (PCr) and adenylate in the isovolumic acetate-perfused rat heart. Various concentrations of PCr and ATP were induced by prior perfusion with 2 deoxy-D-glucose in the presence of insulin. The apparent rate constant (k(f)) and the forward CK flux were measured under metabolic and contractile steady state by progressive saturation-transfer 31P nuclear magnetic resonance (NMR). At high adenylate content CK flux was constant for a twofold reduction in PCr concentration ([PCr]); CK flux was 6.3 +/- 0.6 mM/s (vs. 6.5 +/- 0.2 mM/s in control) because of a doubling of k(f). Although, at the lowest ATP concentration and [PCr], CK flux was reduced by 50%, it nevertheless always remained higher than ATP synthesis estimated by parallel oxygen consumption measurement. NMR-measured flux was compared with the flux computed under the hypothesis of CK equilibrium. CK flux could not be fully predicted by the concentrations of CK metabolites. This is discussed in terms of metabolite and CK isozyme compartmentation.

Asunto(s)

Adenosina Trifosfato/metabolismo , Creatina Quinasa/metabolismo , Corazón/fisiología , Miocardio/metabolismo , Fosfocreatina/metabolismo , Animales , Desoxiglucosa/metabolismo , Corazón/efectos de los fármacos , Insulina/farmacología , Cinética , Espectroscopía de Resonancia Magnética , Masculino , Modelos Cardiovasculares , Modelos Químicos , Contracción Miocárdica , Perfusión , Ratas , Ratas Wistar , Análisis de Regresión

RESUMEN

Carotenoid synthesis in Escherichia coli, when transformed with plasmid containing a carotenoid gene cluster from Erwinia herbicola (pPL376), is regulated by RpoS. When the plasmid was transformed into E. coli mutants that were oxyR minus, the intracellular carotenoid concentration dramatically increased from that observed in an oxyR plus allele. The higher carotenoid concentration in these mutants correlated with an increase in rpoS transcription as indicated by beta-galactosidase activity from a rpoS::lacZ promoter fusion. This indication of a higher concentration of carotenoids correlated with an increased resistance to hydrogen peroxide and near-ultraviolet radiation (310-400 nm; near-UV).

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Carotenoides/biosíntesis , Proteínas de Unión al ADN , Erwinia/genética , Escherichia coli/genética , Proteínas Represoras/fisiología , Factor sigma/metabolismo , Factores de Transcripción/fisiología , Proteínas Bacterianas/genética , Carotenoides/genética , Erwinia/metabolismo , Escherichia coli/efectos de la radiación , Proteínas de Escherichia coli , Estrés Oxidativo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Factor sigma/genética , Espectrofotometría Atómica , Transcripción Genética , Rayos Ultravioleta

RESUMEN

Since myocardial contractility relies on the continuous balance of ATP synthesis and ATP degradation, a dynamic estimation of the high energy phosphates (HEP) turnover by magnetization transfer 31P NMR spectroscopy appears to be a more valuable index of heart function than the static evaluation of HEP concentration. The theory of the main magnetization transfer techniques (saturation transfer, inversion transfer) is described as well as a critical evaluation of their application to the determination of creatine kinase (CK) fluxes in myocardium. The determinants of CK flux in vivo is evaluated (total CK activity, isozymic CK expression and concentrations of CK substrates and products). CK flux is shown to vary in relation to contractility during short term stress (hypoxia, ischemia) and in long term adaptation or pathology of the myocardium. The dynamic estimation of energetic flux in vivo is proposed as a non-invasive tool of diagnosis in myocardial pathologies.

Asunto(s)

Bioquímica/métodos , Creatina Quinasa/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Miocardio/enzimología , Animales , Humanos , Hipoxia/enzimología , Imagen por Resonancia Magnética/métodos , Isquemia Miocárdica/enzimología

RESUMEN

The effects of chronic hypobaric hypoxia (CHH, 28 days, simulated altitude 5,500 m) on the cardiac expression of myosin heavy chain (MHC) and creatine kinase (CK) was studied in rat left (LV) and right (RV) ventricle. To separate the effects of hypoxia from its associated perturbations, anorexia and pulmonary hypertension (resulting in RV hypertrophy), CHH animals were compared with normoxic controls (C) and with rats restricted in food supply (pair fed, PF). In RV, the increased proportion of beta-MHC in CHH (20 +/- 3%) vs. C (7 +/- 2%, P < 0.01) and vs. PF (12 +/- 2%, P < 0.05) rats was mainly attributed to hypertension. In contrast, the higher beta-MHC of CHH (23 +/- 2%) vs. C (13 +/- 2%, P < 0.05) in LV was mainly ascribed to anorexia (PF = 21 +/- 3%, not significant). A major contribution of anorexia was also evidenced in the isozymic profile of CK; anorexia accounted for a 25% decrease in mito-CK specific activity in LV, whereas hypertension partly accounted for the threefold increase in BB-CK in RV. CHH specifically induced a twofold rise in LV BB-CK. This suggests that both the expression of slow myosin, improving the economy of contraction, and the changes in CK isozymic profile could provide a biochemical basis for the CHH resistance to ischemia.

Asunto(s)

Anorexia/fisiopatología , Creatina Quinasa/biosíntesis , Citocromos/metabolismo , Hipertensión/fisiopatología , Hipoxia/fisiopatología , Miocardio/metabolismo , Cadenas Pesadas de Miosina/biosíntesis , Aclimatación , Adenilato Quinasa/metabolismo , Altitud , Análisis de Varianza , Animales , Enfermedad Crónica , Complejo IV de Transporte de Electrones/metabolismo , Ventrículos Cardíacos , Técnicas In Vitro , Isoenzimas , Masculino , Ratas , Ratas Wistar , Triyodotironina/sangre

RESUMEN

We propose a simple mathematical model and a practical approach for evaluating the flux constant and the absolute value of flux in the citric acid cycle in perfused organs by 13C-NMR and 1H-NMR spectroscopy. We demonstrate that 13C-NMR glutamate spectra are independent of the relative sizes of the mitochondrial and cytosolic compartments and the exchange rates of glutamates, unless there is a difference in 13C chemical shifts of glutamate carbons between the two compartments. Wistar rat hearts (five beating and four KCl-arrested hearts) were aerobically perfused with 100% enriched [2-(13)C]acetate and the kinetics of glutamate carbon labeling from perchloric acid extracts were studied at various perfusion times. Under our experimental conditions, the citric acid cycle flux constant, which represents the fraction of glutamate in exchange with the citric acid cycle per unit time, is about 0.350 +/- 0.003 min(-1) for beating hearts and 0.0741 +/- 0.004 min(-1) for KCl-arrested hearts. The absolute values of the citric acid flux for beating hearts and for KCl-arrested hearts are 1.06 +/- 0.06 micromol x min(-1) x mg(-1) and 0.21 +/- 0.02 micromol x min(-1) x g(-1), respectively. The fraction of unlabeled acetate determined from the proton signal of the methyl group is small and essentially the same in beating and arrested hearts (7.4 +/- 1.7% and 8.8 +/- 2.1%, respectively). Thus, the large difference in the Glu C2/C4 between beating and arrested hearts is not due to the important contribution from anaplerotic sources in arrested hearts but simply to a substantial difference in citric acid cycle fluxes. Our model fits the experimental data well, indicating a fast exchange between 2-oxoglutarate and glutamate in the mitochondria of rat hearts. Analysis of the flux constant, calculated from the half-time of glutamate C4 labeling given in the literature, allows for a comparison of the citric acid flux for various working conditions in different animal species.

Asunto(s)

Ciclo del Ácido Cítrico/fisiología , Corazón/fisiología , Modelos Biológicos , Ácido Acético/metabolismo , Animales , Isótopos de Carbono , Compartimento Celular , Ácido Glutámico , Hidrógeno , Espectroscopía de Resonancia Magnética , Masculino , Perfusión , Ratas , Ratas Wistar

RESUMEN

The regulation of cardiac L-type Ca2- current (Ica) and contraction by dihydropyridine antagonists and beta-adrenergic receptor agonists has been the subject of numerous studies over the last decade. However, little is known on the crosstalk between these two regulatory pathways. For instance, a fundamental question that remains unanswered is: does activation of the beta-adrenergic receptors modify the sensitivity of the myocardium to dihydropyridine agonists? To answer this question, we examined in the present study how activation of the beta-adrenergic receptors modifies the effects of nifedipine on the mechanical and energetic parameters of the isolated perfused rat heart. Activation of the beta-adrenergic receptors was achieved by perfusing the hearts with isoprenaline, a non-selective beta-adrenergic receptor agonist, and could be reduced by atenolol, a beta-adrenergic receptor antagonist. To examine possible alterations during hypertension in the sensitivity of the hearts to the drug, tested, the study was performed in both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive animals (SHR). While 0.1 microM nifedipine reduced left ventricular pressure (LVP) by 36% and 34% in WKY and SHR rats, respectively, under basal conditions, its effects became negligible in both groups of rats after stimulation of the hearts with 0.1 microM isoprenaline. Addition of 1 microM atenolol in the presence of isoprenaline restored the inhibitory effect of nifedipine to control values in both WKY and SHR rats. Additional experiments were performed in isolated ventricular myocytes from WKY rats using the whole-cell patch-clamp technique. The inhibitory effects of 0.1 to 1 microM nifedipine were significantly larger on basal Ica than after the current had been previously elevated by 0.1 microM isoprenaline. Addition of 1 microM atenolol in the presence of isoprenaline partially restored the inhibitory effect of nifedipine on Ica. Our results demonstrate a reduced sensitivity of the heart muscle to nifedipine during activation of beta 1-adrenergic receptors. This effect is partly explained by a reduced inhibitory effect of nifedipine on Ic during activation of cAMP-dependent phosphorylation.

Asunto(s)

Canales de Calcio/metabolismo , Contracción Miocárdica/efectos de los fármacos , Nifedipino/farmacología , Receptores Adrenérgicos beta/metabolismo , Simpatomiméticos/farmacología , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta , Animales , Atenolol/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Células Cultivadas , Dihidropiridinas/antagonistas & inhibidores , Ventrículos Cardíacos/citología , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , Técnicas In Vitro , Isoproterenol/farmacología , Masculino , Contracción Miocárdica/fisiología , Reperfusión Miocárdica , Técnicas de Placa-Clamp , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores Adrenérgicos beta/efectos de los fármacos , Simpaticolíticos/farmacología , Función Ventricular

RESUMEN

AMP degradation is studied in two models of the Langendorff-perfused rat heart which generate a large release of purines: the 2-deoxy-D-glucose (2DG)-perfused heart and the anoxic heart. In the 2DG model, mitochondrial energy generation is quasi-normal, despite a very low ATP concentration. Furthermore, inorganic phosphate (Pi) concentration is low, an important difference with anoxia where Pi is very high, up to 82 mM. Coronary release of purines is measured by high performance liquid chromatography, and myocardial metabolite content by 31P nuclear magnetic resonance spectroscopy. In the 2DG-perfused hearts with glucose or acetate, the purine release consists nearly exclusively of inosine [up to 130 nmol/(min x gww)] while adenosine is less than 1 nmol/(min x gww). A possible interpretation is that AMP degradation proceeds mainly through deamination to inosine monophosphate by AMP deaminase (the IMP pathway). In contrast, the purine release in anoxia (100% N2) contains comparable quantities of adenosine and inosine [respectively 30 and 20 nmol/(min x gww)], indicating that part of AMP is dephosphorylated directly to adenosine. Comparison with the 2DG model suggests that the release of adenosine in the anoxic heart is a result of inhibition of AMP deaminase by Pi.

Asunto(s)

Adenosina Monofosfato/metabolismo , Adenosina/metabolismo , Desoxiglucosa/farmacología , Inosina/metabolismo , Miocardio/metabolismo , Animales , Antimetabolitos/metabolismo , Antimetabolitos/farmacología , Desoxiglucosa/metabolismo , Glucosa/farmacología , Glucosa-6-Fosfato/análogos & derivados , Glucosa-6-Fosfato/biosíntesis , Corazón/fisiología , Hemodinámica , Hipoxia/metabolismo , Técnicas In Vitro , Masculino , Perfusión , Ratas , Ratas Sprague-Dawley , Acetato de Sodio/farmacología

RESUMEN

In Escherichia coli, fur mutants that constitutively express their native iron chelating agent, enterobactin, are significantly more sensitive to near-UV radiation (NUV) than wild type, An entA mutant, which is incapable of synthesizing enterobactin, is equal to wild type in resistance to NUV irradiation. However, the addition of Fe+3 enterobactin but not AI+3 enterobactin to entA cell suspensions just prior to irradiation results in an increased sensitivity to NUV irradiation. A fes mutant, which is unable to reduce and release iron from enterobactin, is significantly more sensitive to NUV irradiation than wild type. The addition of nontoxic levels of H2O2 (5 microM) just prior to irradiation significantly increases sensitivity of both fur and fes mutants. These results suggest that one mechanism by which NUV irradiation leads to cell lethality is by creating a transient iron overload, producing very favorable conditions for the production of highly deleterious free radicals through a variety of mechanisms that lead to oxidative stress and DNA damage including lethal and mutagenic lesions. These results are consistent with the hypothesis that enterobactin is an endogenous chromophore for NUV and contributes to cell lethality via the destruction of its ligand, releasing Fe+2 into the cytoplasm to catalyze the production of highly reactive hydroxyl radicals and other toxic oxygen species via the Haber-Weiss reaction.

Asunto(s)

Enterobactina/metabolismo , Escherichia coli/metabolismo , Escherichia coli/efectos de la radiación , Hierro/metabolismo , Escherichia coli/genética , Mutación , Tolerancia a Radiación , Especies Reactivas de Oxígeno/metabolismo , Sideróforos/metabolismo , Rayos Ultravioleta

RESUMEN

This article is a review on the organization and function of myofibrillar creatine kinase in striated muscle. The first part describes myofibrillar creatine kinase as an integral structural part of the complex organization of myofibrils in striated muscle. The second part considers the intrinsic biochemical and mechanical properties of myofibrils and the functional coupling between myofibrillar CK and myosin ATPase. Skinned fiber studies have been developed to evidence this functional coupling and the consequences for cardiac contraction. The data show that creatine kinase in myofibrils is effective enough to sustain normal tension and relaxation, normal Ca sensitivity and kinetic characteristics. Moreover, the results suggest that myofibrillar creatine kinase is essential in maintaining adequate ATP/ADP ratio in the vicinity of myosin ATPase active site to prevent dysfunctioning of this enzyme. Implications for the physiology and physiopathology of cardiac muscle are discussed.

Asunto(s)

Creatina Quinasa/metabolismo , Contracción Miocárdica/fisiología , Miofibrillas/enzimología , Citoesqueleto de Actina/enzimología , Adenosina Trifosfato/farmacología , Animales , Fenómenos Biomecánicos , Metabolismo Energético , Humanos , Técnicas In Vitro , Contracción Miocárdica/efectos de los fármacos , Isquemia Miocárdica/fisiopatología , Miosinas/metabolismo , Sarcómeros/fisiología