RESUMEN
AIM: Production of reactive oxygen species (ROS) in skeletal muscle is markedly increased during exercise and may be essential for exercise adaptation. We, therefore, investigated the effects of infusion with the antioxidant N-acetylcysteine (NAC) on exercise-induced activation of signalling pathways and genes involved in exercise adaptation in human skeletal muscle. METHODS: Subjects completed two exercise tests, 7 days apart, with saline (control, CON) or NAC infusion before and during exercise. Exercise tests comprised of cycling at 71% VO(2peak) for 45 min, and then 92% VO(2peak) to fatigue, with vastus lateralis biopsies at pre-infusion, after 45-min cycling and at fatigue. RESULTS: Analysis was conducted on the mitogen-activated protein kinase signalling pathways, demonstrating that NAC infusion blocked the exercise-induced increase in JNK phosphorylation, but not ERK1/2, or p38 MAPK. Nuclear factor-κB p65 phosphorylation was unaffected by exercise; however, it was reduced in NAC at fatigue by 14% (P < 0.05) compared with pre-infusion. Analysis of exercise and/or ROS-sensitive genes demonstrated that exercise-induced mRNA expression is ROS dependent of MnSOD, but not PGC-1α, interleukin-6, monocyte chemotactic protein-1, or heat-shock protein 70. CONCLUSION: These results suggest that inhibition of ROS attenuates some skeletal muscle cell signalling pathways and gene expression involved in adaptations to exercise.
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Acetilcisteína/administración & dosificación , Antioxidantes/administración & dosificación , Ejercicio Físico , Contracción Muscular , Estrés Oxidativo/efectos de los fármacos , Músculo Cuádriceps/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Adaptación Fisiológica , Adulto , Análisis de Varianza , Ciclismo , Biopsia , Quimiocina CCL2/genética , Estudios Cruzados , Método Doble Ciego , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Humanos , Proteínas I-kappa B/metabolismo , Infusiones Intravenosas , Interleucina-6/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Fatiga Muscular , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Consumo de Oxígeno , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosforilación , Músculo Cuádriceps/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Superóxido Dismutasa/genética , Factores de Tiempo , Factores de Transcripción/genética , Victoria , Adulto JovenRESUMEN
Exercise increases Na(+)-K(+) pump isoform gene expression and elevates muscle reactive oxygen species (ROS). We investigated whether enhanced ROS scavenging induced with the antioxidant N-acetylcysteine (NAC) blunted the increase in Na(+)-K(+) pump mRNA during repeated contractions in human and rat muscle. In experiment 1, well-trained subjects received saline or NAC intravenously prior to and during 45 min cycling. Vastus lateralis muscle biopsies were taken pre-infusion and following exercise. In experiment 2, isolated rat extensor digitorum longus muscles were pre-incubated without or with 10 mm NAC and then rested or stimulated electrically at 60 Hz for 90 s. After 3 h recovery, muscles were frozen. In both experiments, the muscles were analysed for Na(+)-K(+) pump alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) mRNA. In experiment 1, exercise increased alpha(2) mRNA by 1.0-fold (P = 0.03), but alpha(2) mRNA was reduced by 0.40-fold with NAC (P = 0.03). Exercise increased alpha(3), beta(1) and beta(2) mRNA by 2.0- to 3.4-fold (P < 0.05), but these were not affected by NAC (P > 0.32). Neither exercise nor NAC altered alpha(1) or beta(3) mRNA (P > 0.31). In experiment 2, electrical stimulation increased alpha(1), alpha(2) and alpha(3) mRNA by 2.3- to 17.4-fold (P < 0.05), but these changes were abolished by NAC (P > 0.07). Electrical stimulation almost completely reduced beta(1) mRNA but only in the presence of NAC (P < 0.01). Neither electrical stimulation nor NAC altered beta(2) or beta(3) mRNA (P > 0.09). In conclusion, NAC attenuated the increase in Na(+)-K(+) pump alpha(2) mRNA with exercise in human muscle and all alpha isoforms with electrical stimulation in rat muscle. This indicates a regulatory role for ROS in Na(+)-K(+) pump alpha isoform mRNA in mammalian muscle during repeated contractions.
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Acetilcisteína/farmacología , Antioxidantes/farmacología , Ejercicio Físico , Contracción Muscular , Músculo Cuádriceps/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Acetilcisteína/administración & dosificación , Adulto , Animales , Antioxidantes/administración & dosificación , Estudios Cruzados , Método Doble Ciego , Estimulación Eléctrica , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Infusiones Intravenosas , Masculino , Isoformas de Proteínas , Músculo Cuádriceps/enzimología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/genética , Adulto JovenRESUMEN
We aimed to quantify the Na(+)-K(+) exchange occurring during exercise in rat skeletal muscle in vivo. Intracellular Na(+) and K(+) content, Na(+) permeability ((22)Na(+) influx), Na(+)-K(+) pump activity (ouabain-sensitive (86)Rb(+) uptake) and Na(+)-K(+) pump alpha(2) subunit content ([(3)H]ouabain binding) were measured. Six-week-old rats rested (control animals) or performed intermittent running for 10-60 min and were then killed or were killed at 15 or 90 min following 60 min exercise. In the soleus muscle, intracellular Na(+) was 80% higher than in control rats after 60 min exercise, was still elevated (38%) after 15 min rest and returned to control levels after 90 min rest. Intracellular K(+) showed corresponding decreases after 15-60 min exercise, returning to control levels 90 min postexercise. Exercise induced little change in Na(+) and K(+) in the extensor digitorum longus muscle (EDL). In soleus, the exercise-induced rise in Na(+) and reduction in K(+) were augmented by pretreatment with ouabain or by reducing the content of muscular Na(+)-K(+) pumps by prior K(+) depletion of the animals. Fifteen minutes after 60 min exercise, ouabain-sensitive (86)Rb(+) uptake in the soleus was increased by 30% but was unchanged in EDL, and there was no effect of exercise on [(3)H]ouabain binding measured in vitro or in vivo in either muscle. In conclusion, in the soleus, in vivo exercise induces a rise in intracellular Na(+), which reflects the excitation-induced increase in Na(+) influx and leads to augmented Na(+)-K(+) pump activity without apparent change in Na(+)-K(+) pump capacity.
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Contracción Muscular , Músculo Esquelético/enzimología , Esfuerzo Físico , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Sitios de Unión , Bumetanida/farmacología , Permeabilidad de la Membrana Celular , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Masculino , Músculo Esquelético/efectos de los fármacos , Ouabaína/metabolismo , Ouabaína/farmacología , Potasio/metabolismo , Ratas , Ratas Wistar , Radioisótopos de Rubidio , Sodio/metabolismo , Radioisótopos de Sodio , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Factores de TiempoRESUMEN
The Na+ -K+ -ATPase enzyme is vital in skeletal muscle function. We investigated the effects of acute high-intensity interval exercise, before and following high-intensity training (HIT), on muscle Na+ -K+ -ATPase maximal activity, content, and isoform mRNA expression and protein abundance. Twelve endurance-trained athletes were tested at baseline, pretrain, and after 3 wk of HIT (posttrain), which comprised seven sessions of 8 x 5-min interval cycling at 80% peak power output. Vastus lateralis muscle was biopsied at rest (baseline) and both at rest and immediately postexercise during the first (pretrain) and seventh (posttrain) training sessions. Muscle was analyzed for Na+ -K+ -ATPase maximal activity (3-O-MFPase), content ([3H]ouabain binding), isoform mRNA expression (RT-PCR), and protein abundance (Western blotting). All baseline-to-pretrain measures were stable. Pretrain, acute exercise decreased 3-O-MFPase activity [12.7% (SD 5.1), P < 0.05], increased alpha1, alpha2, and alpha3 mRNA expression (1.4-, 2.8-, and 3.4-fold, respectively, P < 0.05) with unchanged beta-isoform mRNA or protein abundance of any isoform. In resting muscle, HIT increased (P < 0.05) 3-O-MFPase activity by 5.5% (SD 2.9), and alpha3 and beta3 mRNA expression by 3.0- and 0.5-fold, respectively, with unchanged Na+ -K+ -ATPase content or isoform protein abundance. Posttrain, the acute exercise induced decline in 3-O-MFPase activity and increase in alpha1 and alpha3 mRNA each persisted (P < 0.05); the postexercise 3-O-MFPase activity was also higher after HIT (P < 0.05). Thus HIT augmented Na+ -K+ -ATPase maximal activity despite unchanged total content and isoform protein abundance. Elevated Na+ -K+ -ATPase activity postexercise may contribute to reduced fatigue after training. The Na+ -K+ -ATPase mRNA response to interval exercise of increased alpha- but not beta-mRNA was largely preserved posttrain, suggesting a functional role of alpha mRNA upregulation.
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Adaptación Fisiológica , Ejercicio Físico/fisiología , Fatiga Muscular , Resistencia Física/fisiología , Músculo Cuádriceps/enzimología , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Adaptación Fisiológica/genética , Inducción Enzimática , Fluoresceínas/metabolismo , Humanos , Isoenzimas/metabolismo , Masculino , Fatiga Muscular/genética , Ouabaína/metabolismo , Resistencia Física/genética , Unión Proteica , ARN Mensajero/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
AIM: This study investigated the effects of endurance training status and sex differences on skeletal muscle Na+,K+-pump mRNA expression, content and activity. METHODS: Forty-five endurance-trained males (ETM), 11 recreationally active males (RAM), and nine recreationally active females (RAF) underwent a vastus lateralis muscle biopsy. Muscle was analysed for Na+,K+-pump alpha1, alpha2, alpha3, beta1, beta2 and beta3 isoform mRNA expression (real-time reverse transcription-polymerase chain reaction), content ([3H]-ouabain-binding site) and maximal activity (3-O-methylfluorescein phosphatase, 3-O-MFPase). RESULTS: ETM demonstrated lower alpha1, alpha3, beta2 and beta3 mRNA expression by 74%, 62%, 70% and 82%, respectively, than RAM (P<0.04). In contrast, [3H]-ouabain binding and 3-O-MFPase activity were each higher in ETM than in RAM, by 16% (P<0.03). RAM demonstrated a 230% and 364% higher alpha3 and beta3 mRNA expression than RAF, respectively (P<0.05), but no significant sex differences were found for alpha1, alpha2, beta1 or beta2 mRNA, [3H]-ouabain binding or 3-O-MFPase activity. No significant correlation was found between years of endurance training and either [3H]-ouabain binding or 3-O-MFPase activity. Significant but weak correlations were found between the number of training hours per week and 3-O-MFPase activity (r=0.31, P<0.02) and between incremental exercise VO2(peak)) and both [3H]-ouabain binding (r=0.33, P<0.01) and 3-O-MFPase activity (r=0.28, P<0.03). CONCLUSIONS: Isoform-specific differences in Na+,K+-pump mRNA expression were found with both training status and sex differences, but only training status influenced Na+,K+-pump content and maximal activity in human skeletal muscle.
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Regulación de la Expresión Génica , Músculo Esquelético/enzimología , Resistencia Física , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Adulto , Análisis de Varianza , Sitios de Unión , Biopsia , Estudios Transversales , Ciclofilinas/genética , Activación Enzimática , Femenino , Humanos , Masculino , Ouabaína/metabolismo , Educación y Entrenamiento Físico , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores Sexuales , ATPasa Intercambiadora de Sodio-Potasio/análisis , Factores de TiempoRESUMEN
We investigated the role of limitations in aerobic metabolism, glycolysis, and membrane excitability for development of high-frequency fatigue in isolated rat soleus muscle. Muscles mounted on force transducers were incubated in buffer bubbled with 5% CO(2) and either 95% O(2) (oxygenated) or 95% N(2) (anoxic) and stimulated at 60 Hz continuously for 30-120 s or intermittently for 120 s. Cyanide (2 mM) and 2-deoxyglucose (10 mM) were used to inhibit aerobic metabolism and both glycolysis and aerobic metabolism, respectively. Excitability was reduced by carbacholine (10 microM), a nicotinic ACh receptor agonist, or ouabain (10 microM), an Na(+)-K(+) pump inhibitor. Membrane excitability was measured by recording M waves. Intracellular Na(+) and K(+) contents and membrane potentials were measured by flame photometry and microelectrodes, respectively. During 120 s of continuous stimulation, oxygenated and anoxic muscles showed the same force loss. In oxygenated muscles, cyanide did not alter force loss for up to 90 s, whereas 2-deoxyglucose increased force loss (by 19-69%; P < 0.01) from 14 s of stimulation. In oxygenated muscles, 60 s of stimulation reduced force, M wave area, and amplitude by 70-90% (P < 0.001). Carbacholine or ouabain increased intracellular Na(+) content (P < 0.001), induced a 7- to 8-mV membrane depolarization (P < 0.001), and accelerated the rate of force loss (by 250-414%) during 30 s of stimulation (P < 0.001). Similar effects were seen with intermittent stimulation. In conclusion, limitations in glycolysis and subsequently also in aerobic metabolism, as well as membrane excitability but not aerobic metabolism alone, appear to play an important role in the development of high-frequency fatigue in isolated rat soleus muscle.
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Membrana Celular/fisiología , Metabolismo Energético/fisiología , Glucólisis/fisiología , Fatiga Muscular/fisiología , Músculo Esquelético/fisiología , Aerobiosis , Animales , Carbacol/farmacología , Metabolismo Energético/efectos de los fármacos , Glucólisis/efectos de los fármacos , Fatiga Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Ouabaína/farmacología , Ratas , Ratas Wistar , Tetania , Factores de TiempoRESUMEN
BACKGROUND AND PURPOSE: In cardiac muscle, BRL 37344, a selective beta3-adrenoceptor agonist, activates the Na+, K+ -pump via NO signalling. This study investigated whether BRL 37344 also activates the Na+, K+ -pump via beta3-adrenoceptors in skeletal muscle. EXPERIMENTAL APPROACH: Isolated rat soleus muscles were incubated between 1 and 60 min in buffer. Intracellular Na+, K+ content and Na+, K+ -pump activity were measured using flame photometry and ouabain-suppressible 86Rb+ uptake, respectively. Additional muscles were mounted on force transducers and stimulated (60 Hz for 2 s) every 10 min. KEY RESULTS: BRL 37344 (10(-8) -10(-5) M) induced a concentration- and time-dependent reduction in intracellular Na+, and increased ouabain-suppressible 86Rb+ uptake by up to 112%. BRL 37344-induced reductions in intracellular Na+ were blocked by the beta1/beta2-adrenoceptor antagonist, nadolol (10(-7) M), and the beta2-adrenoceptor antagonist, ICI 118,551 (10(-7) -10(-5) M), but not by beta3- or beta1-adrenoceptor antagonists, SR 59230A (10(-7) M) and CGP 20712A (10(-7) -10(-5) M), respectively. Another beta3-adrenoceptor agonist, CL 316,243, did not alter intracellular Na+. BRL 37344-induced reductions in intracellular Na+ were not blocked by L-NAME, an NOS inhibitor, or ODQ, a guanylyl cyclase inhibitor. The NO donors, SNP and SNAP, did not alter intracellular Na+. BRL 37344 rapidly recovered force in muscles depressed by high [K+]o, an effect that was blocked by nadolol, but not L-NAME. CONCLUSIONS AND IMPLICATIONS: In rat soleus muscle, the beta3-adrenoceptor agonist BRL 37344 stimulated the Na+, K+ -pump via beta2-adrenoceptors. A more selective beta3-adrenoceptor agonist did not affect Na+, K+ homeostasis in skeletal muscle. NO did not seem to mediate Na+, K+ -pump stimulation in skeletal muscle.
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Agonistas Adrenérgicos beta/farmacología , Receptores Adrenérgicos beta 3/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Ratas , Ratas Wistar , Receptores Adrenérgicos beta 2/fisiologíaRESUMEN
This study investigated effects of prolonged submaximal exercise on Na+-K+-ATPase mRNA and protein expression, maximal activity, and content in human skeletal muscle. We also investigated the effects on mRNA expression of the transcription initiator gene, RNA polymerase II (RNAP II), and key genes involved in protein translation, eukaryotic initiation factor-4E (eIF-4E) and 4E-binding protein 1 (4E-BP1). Eleven subjects (6 men, 5 women) cycled at 75.5% (SD 4.8%) peak O2 uptake and continued until fatigue. A vastus lateralis muscle biopsy was taken at rest, fatigue, and 3 and 24 h postexercise. We analyzed muscle for Na+-K+-ATPase alpha1, alpha2, alpha3, beta1, beta2, and beta3, as well for RNAP II, eIF-4E, and 4E-BP1 mRNA expression by real-time RT-PCR and Na+-K+-ATPase isoform protein abundance using immunoblotting. Muscle homogenate maximal Na+-K+-ATPase activity was determined by 3-O-methylfluorescein phosphatase activity and Na+-K+-ATPase content by [3H]ouabain binding. Cycling to fatigue [54.5 (SD 20.6) min] immediately increased alpha3 (P = 0.044) and beta2 mRNA (P = 0.042) by 2.2- and 1.9-fold, respectively, whereas alpha1 mRNA was elevated by 2.0-fold at 24 h postexercise (P = 0.036). A significant time main effect was found for alpha3 protein abundance (P = 0.046). Exercise transiently depressed maximal Na+-K+-ATPase activity (P = 0.004), but Na+-K+-ATPase content was unaltered throughout recovery. Exercise immediately increased RNAP II mRNA by 2.6-fold (P = 0.011) but had no effect on eIF-4E and 4E-BP1 mRNA. Thus a single bout of prolonged submaximal exercise induced isoform-specific Na+-K+-ATPase responses, increasing alpha1, alpha3, and beta2 mRNA but only alpha3 protein expression. Exercise also increased mRNA expression of RNAP II, a gene initiating transcription, but not of eIF-4E and 4E-BP1, key genes initiating protein translation.
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Ejercicio Físico/fisiología , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adulto , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Masculino , Músculo Esquelético/enzimología , Ouabaína/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
This study investigated the effects of electrical stimulation on Na+-K+-ATPase isoform mRNA, with the aim to identify factors modulating Na+-K+-ATPase mRNA in isolated rat extensor digitorum longus (EDL) muscle. Interventions designed to mimic exercise-induced increases in intracellular Na+ and Ca2+ contents and membrane depolarization were examined. Muscles were mounted on force transducers and stimulated with 60-Hz 10-s pulse trains producing tetanic contractions three times at 10-min intervals. Ouabain (1.0 mM, 120 min), veratridine (0.1 mM, 30 min), and monensin (0.1 mM, 30 min) were used to increase intracellular Na+ content. High extracellular K+ (13 mM, 60 min) and the Ca2+ ionophore A-23187 (0.02 mM, 30 min) were used to induce membrane depolarization and elevated intracellular Ca2+ content, respectively. Muscles were analyzed for Na+-K+-ATPase alpha1-alpha3 and beta1-beta3 mRNA (real-time RT-PCR). Electrical stimulation had no immediate effect on Na+-K+-ATPase mRNA; however at 3 h after stimulation, it increased alpha1, alpha2, and alpha3 mRNA by 223, 621, and 892%, respectively (P = 0.010), without changing beta mRNA. Ouabain, veratridine, and monensin increased intracellular Na+ content by 769, 724, and 598%, respectively (P = 0.001) but did not increase mRNA of any isoform. High intracellular K+ concentration elevated alpha1 mRNA by 160% (P = 0.021), whereas A-23187 elevated alpha3 mRNA by 123% (P = 0.035) but reduced beta1 mRNA by 76% (P = 0.001). In conclusion, electrical stimulation induced subunit-specific increases in Na+-K+-ATPase mRNA in isolated rat EDL muscle. Furthermore, Na+-K+-ATPase mRNA appears to be regulated by different stimuli, including cellular changes associated with membrane depolarization and increased intracellular Ca2+ content but not increased intracellular Na+ content.
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Regulación Enzimológica de la Expresión Génica/fisiología , Canales Iónicos/fisiología , Músculo Esquelético/enzimología , ARN Mensajero/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Calcimicina/farmacología , Calcio/metabolismo , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Ionóforos/farmacología , Isoenzimas/metabolismo , Masculino , Monensina/farmacología , Husos Musculares/fisiología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Ouabaína/farmacología , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sodio/metabolismo , Sodio/fisiología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/genética , Veratridina/farmacologíaRESUMEN
We investigated whether depressed muscle Na(+)-K(+)-ATPase activity with exercise reflected a loss of Na(+)-K(+)-ATPase units, the time course of its recovery postexercise, and whether this depressed activity was related to increased Na(+)-K(+)-ATPase isoform gene expression. Fifteen subjects performed fatiguing, knee extensor exercise at approximately 40% maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue, 3 h, and 24 h postexercise and analyzed for maximal Na(+)-K(+)-ATPase activity via 3-O-methylfluorescein phosphatase (3-O-MFPase) activity, Na(+)-K(+)-ATPase content via [(3)H]ouabain binding sites, and Na(+)-K(+)-ATPase alpha(1)-, alpha(2)-, alpha(3)-, beta(1)-, beta(2)- and beta(3)-isoform mRNA expression by real-time RT-PCR. Exercise [352 (SD 267) s] did not affect [(3)H]ouabain binding sites but decreased 3-O-MFPase activity by 10.7 (SD 8)% (P < 0.05), which had recovered by 3 h postexercise, without further change at 24 h. Exercise elevated alpha(1)-isoform mRNA by 1.5-fold at fatigue (P < 0.05). This increase was inversely correlated with the percent change in 3-O-MFPase activity from rest to fatigue (%Delta3-O-MFPase(rest-fatigue)) (r = -0.60, P < 0.05). The average postexercise (fatigue, 3 h, 24 h) alpha(1)-isoform mRNA was increased 1.4-fold (P < 0.05) and approached a significant inverse correlation with %Delta3-O-MFPase(rest-fatigue) (r = -0.56, P = 0.08). Exercise elevated alpha(2)-isoform mRNA at fatigue 2.5-fold (P < 0.05), which was inversely correlated with %Delta3-O-MFPase(rest-fatigue) (r = -0.60, P = 0.05). The average postexercise alpha(2)-isoform mRNA was increased 2.2-fold (P < 0.05) and was inversely correlated with the %Delta3-O-MFPase(rest-fatigue) (r = -0.68, P < 0.05). Nonsignificant correlations were found between %Delta3-O-MFPase(rest-fatigue) and other isoforms. Thus acute exercise transiently decreased Na(+)-K(+)-ATPase activity, which was correlated with increased Na(+)-K(+)-ATPase gene expression. This suggests a possible signal-transduction role for depressed muscle Na(+)-K(+)-ATPase activity with exercise.
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Ejercicio Físico , Fatiga/enzimología , Músculo Esquelético/enzimología , ARN Mensajero/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adulto , Volumen Sanguíneo , Femenino , Humanos , Masculino , Concentración Osmolar , Potasio/sangre , Factores de TiempoRESUMEN
The production of reactive oxygen species in skeletal muscle is linked with muscle fatigue. This study investigated the effects of the antioxidant compound N-acetylcysteine (NAC) on muscle cysteine, cystine, and glutathione and on time to fatigue during prolonged, submaximal exercise in endurance athletes. Eight men completed a double-blind, crossover study, receiving NAC or placebo before and during cycling for 45 min at 71% peak oxygen consumption (VO2 peak) and then to fatigue at 92% VO2 peak. NAC was intravenously infused at 125 mg.kg(-1).h(-1) for 15 min and then at 25 mg.kg(-1).h(-1) for 20 min before and throughout exercise. Arterialized venous blood was analyzed for NAC, glutathione status, and cysteine concentration. A vastus lateralis biopsy was taken preinfusion, at 45 min of exercise, and at fatigue and was analyzed for NAC, total glutathione (TGSH), reduced glutathione (GSH), cysteine, and cystine. Time to fatigue at 92% VO2 peak was reproducible in preliminary trials (coefficient of variation 5.6 +/- 0.6%) and with NAC was enhanced by 26.3 +/- 9.1% (NAC 6.4 +/- 0.6 min vs. Con 5.3 +/- 0.7 min; P <0.05). NAC increased muscle total and reduced NAC at both 45 min and fatigue (P <0.005). Muscle cysteine and cystine were unchanged during Con, but were elevated above preinfusion levels with NAC (P <0.001). Muscle TGSH (P <0.05) declined and muscle GSH tended to decline (P=0.06) during exercise. Both were greater with NAC (P <0.05). Neither exercise nor NAC affected whole blood TGSH. Whereas blood GSH was decreased and calculated oxidized glutathione increased with exercise (P <0.05), both were unaffected by NAC. In conclusion, NAC improved performance in well-trained individuals, with enhanced muscle cysteine and GSH availability a likely mechanism.
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Acetilcisteína/administración & dosificación , Cisteína/farmacocinética , Glutatión/farmacocinética , Fatiga Muscular/fisiología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Resistencia Física/fisiología , Esfuerzo Físico/fisiología , Adulto , Disponibilidad Biológica , Estudios Cruzados , Método Doble Ciego , Prueba de Esfuerzo , Humanos , Infusiones Intravenosas , Masculino , Fatiga Muscular/efectos de los fármacos , Resistencia Física/efectos de los fármacos , Esfuerzo Físico/efectos de los fármacos , Aptitud Física/fisiologíaRESUMEN
Characterization of expression of, and consequently also the acute exercise effects on, Na(+),K(+)-ATPase isoforms in human skeletal muscle remains incomplete and was therefore investigated. Fifteen healthy subjects (eight males, seven females) performed fatiguing, knee extensor exercise at approximately 40% of their maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue and 3 and 24 h postexercise, and analysed for Na(+),K(+)-ATPase alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) mRNA and crude homogenate protein expression, using Real-Time RT-PCR and immunoblotting, respectively. Each individual expressed gene transcripts and protein bands for each Na(+),K(+)-ATPase isoform. Each isoform was also expressed in a primary human skeletal muscle cell culture. Intense exercise (352 +/- 69 s; mean +/-s.e.m.) immediately increased alpha(3) and beta(2) mRNA by 2.4- and 1.7-fold, respectively (P < 0.05), whilst alpha(1) and alpha(2) mRNA were increased by 2.5- and 3.5-fold at 24 h and 3 h postexercise, respectively (P < 0.05). No significant change occurred for beta(1) and beta(3) mRNA, reflecting variable time-dependent responses. When the average postexercise value was contrasted to rest, mRNA increased for alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) isoforms, by 1.4-, 2.2-, 1.4-, 1.1-, 1.0- and 1.0-fold, respectively (P < 0.05). However, exercise did not alter the protein abundance of the alpha(1)-alpha(3) and beta(1)-beta(3) isoforms. Thus, human skeletal muscle expresses each of the Na(+),K(+)-ATPase alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) isoforms, evidenced at both transcription and protein levels. Whilst brief exercise increased Na(+),K(+)-ATPase isoform mRNA expression, there was no effect on isoform protein expression, suggesting that the exercise challenge was insufficient for muscle Na(+),K(+)-ATPase up-regulation.
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Ejercicio Físico/fisiología , Isoenzimas/genética , Músculo Esquelético/fisiología , ATPasa Intercambiadora de Sodio-Potasio/genética , Adulto , Células Cultivadas , Femenino , Humanos , Masculino , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/enzimología , Músculo Esquelético/citología , ARN Mensajero/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
The AMP-activated protein kinase (AMPK) cascade has been linked to many of the acute effects of exercise on skeletal muscle substrate metabolism, as well as to some of the chronic training-induced adaptations. We determined the effect of 3 wk of intensified training (HIT; 7 sessions of 8 x 5 min at 85% Vo2 peak) in skeletal muscle from well-trained athletes on AMPK responsiveness to exercise. Rates of whole body substrate oxidation were determined during a 90-min steady-state ride (SS) pre- and post-HIT. Muscle metabolites and AMPK signaling were determined from biopsies taken at rest and immediately after exercise during the first and seventh HIT sessions, performed at the same (absolute) pre-HIT work rate. HIT decreased rates of whole body carbohydrate oxidation (P < 0.05) and increased rates of fat oxidation (P < 0.05) during SS. Resting muscle glycogen and its utilization during intense exercise were unaffected by HIT. However, HIT induced a twofold decrease in muscle [lactate] (P < 0.05) and resulted in tighter metabolic regulation, i.e., attenuation of the decrease in the PCr/(PCr + Cr) ratio and of the increase in [AMPfree]/ATP. Resting activities of AMPKalpha1 and -alpha2 were similar post-HIT, with the magnitude of the rise in response to exercise similar pre- and post-HIT. AMPK phosphorylation at Thr172 on both the alpha1 and alpha2 subunits increased in response to exercise, with the magnitude of this rise being similar post-HIT. Acetyl-coenzyme A carboxylase-beta phosphorylation was similar at rest and, despite HIT-induced increases in whole body rates of fat oxidation, did not increase post-HIT. Our results indicate that, in well-trained individuals, short-term HIT improves metabolic control but does not blunt AMPK signaling in response to intense exercise.
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Acidosis/enzimología , Ejercicio Físico/fisiología , Complejos Multienzimáticos/metabolismo , Músculo Esquelético/enzimología , Aptitud Física/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Acetil-CoA Carboxilasa/metabolismo , Adulto , Análisis de Varianza , Glucógeno/metabolismo , Humanos , Ácido Láctico/metabolismo , Consumo de Oxígeno/fisiología , Fosforilación , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVE: To determine the outcome, pattern(s) of failure, and optimal treatment volume in Stage IIIC endometrial carcinoma patients treated with surgery and postoperative radiation therapy (RT). METHODS: Between 1983 and 1998, 30 Stage IIIC endometrial carcinoma patients were treated with primary surgery and postoperative RT at the University of Chicago. All underwent total abdominal hysterectomy, bilateral salpingo-oophorectomy, sampling of pelvic lymph nodes (PLN), and peritoneal cytology. All were noted to have PLN involvement. Para-aortic lymph nodes (PALN) were sampled in 26 cases, and were positive in 14 cases (54%). Twenty women received whole-pelvic RT (WPRT) and 10 (WPRT), plus paraortic RT (extended-field RT, EFRT). One EFRT patient also underwent concomitant whole-abdominal RT (WART). Adjuvant vaginal brachytherapy (VB) was delivered in 10, chemotherapy in 5, and hormonal therapy in 7 patients. RESULTS: At a median follow-up of 32 months, the actuarial 5-year disease-free and cause-specific survivals of the entire group were 33.9% and 55.8%, respectively. Overall, 16 women (53%) relapsed. Sites of failure included the pelvis (23%), abdomen (13%), PALN (13%), and distant (40%). Of the 7 pelvic failures, 4 were vaginal (3 vaginal only). Patients treated with VB had a trend to a lower vaginal recurrence rate (0/10 vs. 4/20, p = 0.12) than those not receiving VB. All 4 PALN failures were in women treated with WPRT (2 negative, 1 unsampled, and 1 positive PALN). None of the 10 EFRT patients (2 negative, 8 positive PALN) recurred in the PALN. No patient developed an isolated abdominal recurrence. Two patients developed significant RT sequelae: chronic diarrhea in 1 patient treated with WPRT and VB, and small bowel obstruction in 1 patient treated with EFRT. CONCLUSION: FIGO Stage IIIC disease comprises a small percentage of endometrial carcinoma patients but carries a poor prognosis. Our failure pattern suggests that the optimal adjuvant RT volume is EFRT, even in women with negative PALN sampling. VB should also be administered to improve local control. The low rate of abdominal recurrence does not support the routine use of WART in these women. Given the predominance of failure in distant sites, attention should be focused on the development of systemic chemotherapy protocols.
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Adenocarcinoma/radioterapia , Neoplasias Endometriales/radioterapia , Histerectomía , Ovariectomía , Radioterapia Adyuvante , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adenocarcinoma/cirugía , Adenocarcinoma de Células Claras/tratamiento farmacológico , Adenocarcinoma de Células Claras/mortalidad , Adenocarcinoma de Células Claras/patología , Adenocarcinoma de Células Claras/radioterapia , Adenocarcinoma de Células Claras/cirugía , Adulto , Anciano , Antineoplásicos Hormonales/uso terapéutico , Braquiterapia , Quimioterapia Adyuvante , Chicago/epidemiología , Terapia Combinada , Cistadenocarcinoma Papilar/tratamiento farmacológico , Cistadenocarcinoma Papilar/mortalidad , Cistadenocarcinoma Papilar/patología , Cistadenocarcinoma Papilar/radioterapia , Cistadenocarcinoma Papilar/cirugía , Supervivencia sin Enfermedad , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/mortalidad , Neoplasias Endometriales/patología , Neoplasias Endometriales/cirugía , Femenino , Estudios de Seguimiento , Humanos , Tablas de Vida , Metástasis Linfática , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia , Estadificación de Neoplasias , Estudios Retrospectivos , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Antibiotic susceptibilities of Pasteurella sp, Haemophilus pleuropneumoniae, and Staphylococcus aureus isolates were determined. The combination of sodium sulbactam, a beta-lactamase inhibitor, and ampicillin had a synergistic effect against all ampicillin-resistant pathogens, rendering them susceptible to ampicillin. Studies of cell-free beta-lactamase from Pasteurella and Haemophilus isolates confirmed the presence of a constitutive penicillinase. Inhibitory concentrations of sulbactam-ampicillin were bactericidal, as demonstrated by killing curves. Ampicillin-resistant Pasteurella and Haemophilus isolates did not develop resistance to sulbactam-ampicillin when passed as many as 8 times in the presence of sublethal concentrations of sulbactam-ampicillin. The in vitro synergistic activity of sulbactam-penicillin also was seen in an in vivo synergistic response in mice challenge exposed to an ampicillin-resistant P haemolytica.