RESUMO
To better understand the mechanisms underlying the pulmonary O(2) uptake (V(O(2P))) slow component during high-intensity exercise, we used (31)P magnetic resonance spectroscopy, gas exchange, surface electromyography and near-infrared spectroscopy measurements to examine the potential relationship between the slow components of V(O(2P)) and phosphocreatine (PCr), muscle recruitment and tissue oxygenation in endurance-trained athletes and sedentary subjects. Specifically, six endurance-trained and seven sedentary subjects performed a dynamic high-intensity exercise protocol during 6 min at an exercise intensity corresponding to 35-40% of knee-extensor maximal voluntary contraction. The slow component of V(O(2P))(117 ± 60 ml min(-1), i.e. 20 ± 10% of the total response) was associated with a paradoxical PCr resynthesis in endurance-trained athletes (-0.90 ± 1.27 mm, i.e. -12 ± 16% of the total response). Meanwhile, oxygenated haemoglobin increased throughout the second part of exercise and was significantly higher at the end of exercise compared with the value at 120 s (P < 0.05), whereas the integrated EMG was not significantly changed throughout exercise. In sedentary subjects, a slow component was simultaneously observed for V(O(2P)) and [PCr] time-dependent changes (208 ± 14 ml min(-1), i.e. 38 ± 18% of the total V(O(2P))response, and 1.82 ± 1.39 mm, i.e. 16 ± 13% of the total [PCr] response), but the corresponding absolute or relative amplitudes were not correlated. The integrated EMG was significantly increased throughout exercise in sedentary subjects. Taken together, our results challenge the hypothesis of a mechanistic link between [PCr] and V(O(2P)) slow components and demonstrate that, as a result of a tighter metabolic control and increased O(2) availability, the [PCr] slow component can be minimized in endurance-trained athletes while the V(O(2P)) slow component occurs.
Assuntos
Exercício Físico/fisiologia , Consumo de Oxigênio/fisiologia , Fosfocreatina/fisiologia , Adulto , Eletromiografia , Feminino , Humanos , Joelho/fisiologia , Espectroscopia de Ressonância Magnética , Masculino , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Fosfocreatina/biossíntese , Resistência Física/fisiologia , Troca Gasosa Pulmonar/fisiologia , Comportamento Sedentário , Adulto JovemRESUMO
If mitochondrial energy availability or oxidative metabolism is altered, patients will suffer from insufficient energy supply Phosphocreatine (PCr) not only acts as an energy carrier, but also acts as an antioxidant and defensive agent to maintain the integrity and stability of the membrane, to maintain ATP homeostasis through regulating mitochondrial respiration. Meanwhile, PCr can enhance calcium balance and reduce morphological pathological changes, ultimately, PCr helps to reduce apoptosis. On the other aspect, the activities of ATP synthase and MitCK play a crucial role in the maintenance of cellular energy metabolic function. It is interesting to note, PCr not only rises the activities of ATP synthase as well as MitCK, but also promotes these two enzymatic reactions. Additionally, PCr can also inhibit mitochondrial permeability transition in a concentration-dependent manner, prevent ROS and CytC from spilling into the cytoplasm, thereby inhibit the release of proapoptotic factors caspase-3 and caspase-9, and eventually, effectively prevent LPS-induced apoptosis of cells. Understandably, PCr prevents the apoptosis caused by abnormal mitochondrial energy metabolism and has a protective role in a non-energy manner. Moreover, recent studies have shown that PCr protects cell survival through PI3K/Akt/eNOS, MAPK pathway, and inhibition of Ang II-induced NF-κB activation. Furthermore, PCr antagonizes oxidative stress through the activation of PI3K/Akt/GSK3b intracellular pathway, PI3K/AKT-PGC1α signaling pathway, while through the promotion of SIRT3 expression to maintain normal cell metabolism. Interestingly, PCr results in delaying the time to enter pathological metabolism through the delayed activation of AMPK pathway, which is different from previous studies, now we propose the hypothesis that the "miRNA-JAK2/STAT3 -CypD pathway" may take part in protecting cells from apoptosis, PCr may be further be involved in the dynamic relationship between CypD and STAT3. Furthermore, we believe that PCr and CypD would be the central link to maintain cell survival and maintain cell stability and mitochondrial repair under the mitochondrial dysfunction caused by oxidative stress. This review provides the modern progress knowledge and views on the molecular mechanism and molecular targets of PCr in a non-energy way.
Assuntos
Sobrevivência Celular/fisiologia , Fosfocreatina/fisiologia , Animais , Sobrevivência Celular/genética , Metabolismo Energético/fisiologia , Humanos , Estresse Oxidativo , Fosfocreatina/genética , Transdução de Sinais/fisiologiaRESUMO
The phenotype of "gene knockout" mice deficient in a creatine kinase isoform sheds new light on the physiological function of the "phosphocreatine circuit."
Assuntos
Creatina Quinase/metabolismo , Fosfocreatina/metabolismo , Animais , Creatina Quinase/genética , Metabolismo Energético , Homeostase , Isoenzimas , Camundongos , Camundongos Mutantes , Modelos Biológicos , Fenótipo , Fosfocreatina/fisiologiaRESUMO
BACKGROUND: The role of the creatine kinase (CK)/phosphocreatine (PCr) energy buffer and transport system in heart remains unclear. Guanidinoacetate-N-methyltransferase-knockout (GAMT-/-) mice represent a new model of profoundly altered cardiac energetics, showing undetectable levels of PCr and creatine and accumulation of the precursor (phospho-)guanidinoacetate (P-GA). To characterize the role of a substantially impaired CK/PCr system in heart, we studied the cardiac phenotype of wild-type (WT) and GAMT-/- mice. METHODS AND RESULTS: GAMT-/- mice did not show cardiac hypertrophy (myocyte cross-sectional areas, hypertrophy markers atrial natriuretic factor and beta-myosin heavy chain). Systolic and diastolic function, measured invasively (left ventricular conductance catheter) and noninvasively (MRI), were similar for WT and GAMT-/- mice. However, during inotropic stimulation with dobutamine, preload-recruitable stroke work failed to reach maximal levels of performance in GAMT-/- hearts (101+/-8 mm Hg in WT versus 59+/-7 mm Hg in GAMT-/-; P<0.05). (31)P-MR spectroscopy experiments showed that during inotropic stimulation, isolated WT hearts utilized PCr, whereas isolated GAMT-/- hearts utilized P-GA. During ischemia/reperfusion, GAMT-/- hearts showed markedly impaired recovery of systolic (24% versus 53% rate pressure product recovery; P<0.05) and diastolic function (eg, left ventricular end-diastolic pressure 23+/-9 in WT and 51+/-5 mm Hg in GAMT-/- during reperfusion; P<0.05) and incomplete resynthesis of P-GA. CONCLUSIONS: GAMT-/- mice do not develop hypertrophy and show normal cardiac function at low workload, suggesting that a fully functional CK/PCr system is not essential under resting conditions. However, when acutely stressed by inotropic stimulation or ischemia/reperfusion, GAMT-/- mice exhibit a markedly abnormal phenotype, demonstrating that an intact, high-capacity CK/PCr system is required for situations of increased cardiac work or acute stress.
Assuntos
Metabolismo Energético/fisiologia , Guanidinoacetato N-Metiltransferase/deficiência , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/etiologia , Fosfocreatina/deficiência , Animais , Cardiomegalia/etiologia , Creatina Quinase/fisiologia , Suscetibilidade a Doenças , Guanidinoacetato N-Metiltransferase/genética , Testes de Função Cardíaca , Hemodinâmica , Camundongos , Camundongos Knockout , Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica/metabolismo , Fosfocreatina/fisiologia , Estresse FisiológicoRESUMO
OBJECTIVE: The neural basis for autistic spectrum disorders is unclear, but abnormalities in the development of limbic areas and of glutamate have been suggested. Proton magnetic resonance spectroscopy ((1)H-MRS) can be used to measure the concentration of brain metabolites. However, the concentration of glutamate/glutamine in brain regions implicated in autistic spectrum disorders has not yet been examined in vivo. METHOD: The authors used (1)H-MRS to investigate the neuronal integrity of the amygdala-hippocampal complex and a parietal control region in adults with autistic spectrum disorders and healthy subjects. RESULTS: People with autistic spectrum disorders had a significantly higher concentration of glutamate/glutamine and creatine/phosphocreatine in the amygdala-hippocampal region but not in the parietal region. CONCLUSIONS: Abnormalities in glutamate/glutamine may partially underpin the pathophysiology of autistic spectrum disorders, and the authors confirm earlier reports that limbic areas are metabolically aberrant in these disorders.
Assuntos
Tonsila do Cerebelo/metabolismo , Transtorno Autístico/diagnóstico , Transtorno Autístico/fisiopatologia , Hipocampo/metabolismo , Espectroscopia de Ressonância Magnética/estatística & dados numéricos , Lobo Parietal/metabolismo , Adulto , Transtorno Autístico/metabolismo , Creatina/metabolismo , Creatina/fisiologia , Feminino , Glutamatos/química , Glutamatos/metabolismo , Glutamatos/fisiologia , Glutamina/metabolismo , Glutamina/fisiologia , Humanos , Testes de Inteligência/estatística & dados numéricos , Masculino , Fosfocreatina/metabolismo , Fosfocreatina/fisiologiaRESUMO
In an effort to investigate the role of creatine kinase and its substrates in malignancy we have tested the effect of cyclocreatine [1-carboxymethyl-2-iminoimidazolidine (CCr)] on the growth of tumor cells in vitro and in vivo. CCr is phosphorylated by creatine kinase to yield a synthetic phosphagen [(N-phosphorylcyclocreatine (CCr approximately P)] with thermodynamic and kinetic properties distinct from those of creatine phosphate. We show that CCr accumulates as CCr approximately P in tumor cells expressing a high level of creatine kinase, and that the accumulation of this phosphagen is detrimental to tumor cell growth. Tumor cell lines expressing a low level of creatine kinase accumulate much less CCr approximately P, and consequently are growth inhibited only at higher concentrations of CCr. When these resistant cells are transfected with a creatine kinase B expression vector, they express creatine kinase, accumulate CCr approximately P, and are growth inhibited. In vivo, in nude mouse xenografts, the rate of growth of a high creatine kinase expressing tumor cell line is inhibited in animals fed 1% CCr. Our results indicate that CCr inhibits the growth of tumor cells in vitro and in vivo.
Assuntos
Antineoplásicos/farmacologia , Creatinina/análogos & derivados , Imidazolidinas , Neoplasias/tratamento farmacológico , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , Transformação Celular Neoplásica , Creatina Quinase/metabolismo , Creatina Quinase/fisiologia , Creatinina/farmacologia , Feminino , Humanos , Isoenzimas , Masculino , Camundongos , Camundongos Nus , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/patologia , Fosfocreatina/análogos & derivados , Fosfocreatina/farmacocinética , Fosfocreatina/fisiologia , Transplante Heterólogo , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/enzimologia , Neoplasias do Colo do Útero/patologiaRESUMO
Charge movement was measured in frog cut twitch fibers with the double Vaseline gap technique. Five manipulations listed below were applied to investigate their effects on the hump component (I gamma) in the ON segments of TEST minus CONTROL current traces. When external Cl-1 was replaced by MeSO3- to eliminate Cl current, I gamma peaked earlier due to a few millivolts shift of the voltage dependence of I gamma kinetics in the negative direction. The Q-V plots in the TEA.Cl and TEA.MeSO3 solutions were well fitted by a sum of two Boltzmann distribution functions. The more steeply voltage-dependent component (Q gamma) had a V approximately 6 mV more negative in the TEA.MeSO3 solution than in the TEA.Cl solution. These voltage shifts were partially reversible. When creatine phosphate in the end pool solution was removed, the I gamma hump disappeared slowly over the course of 20-30 min, partly due to a suppression of Q gamma. The hump reappeared when creatine phosphate was restored. When 0.2-1.0 mM Cd2+ was added to the center pool solution to block inward Ca current, the I gamma hump became less prominent due to a prolongation in the time course of I gamma but not to a suppression of Q gamma. When the holding potential was changed from -90 to -120 mV, the amplitude of I beta was increased, thereby obscuring the I gamma hump. Finally, when a cut fiber was stimulated repetitively, I gamma lost its hump appearance because its time course was prolonged. In an extreme case, a 5-min resting interval was insufficient for a complete recovery of the waveform. In general, a stimulation rate of once per minute had a negligible effect on the shape of I gamma. Of the five manipulations, MeSO3- has the least perturbation on the appearance of I gamma and is potentially a better substitute for Cl- than SO2-(4) in eliminating Cl current if the appearance of the I gamma hump is to be preserved.
Assuntos
Contração Muscular , Músculos/fisiologia , Animais , Anuros , Cádmio/farmacologia , Cloretos/fisiologia , Estimulação Elétrica , Eletrofisiologia , Membranas Intracelulares/metabolismo , Mesilatos/farmacologia , Fosfocreatina/fisiologiaRESUMO
It has been proposed recently that the phosphocreatine (PCr) overshoot (increase above the resting level) during muscle recovery after exercise is caused by a slow decay during this recovery of the direct activation of oxidative phosphorylation taking place during muscle work. In the present article the factors determining the appearance and size of the PCr overshoot are studied using the computer model of oxidative phosphorylation in intact skeletal muscle developed previously. It is demonstrated that the appearance and duration of this overshoot is positively correlated with the value of the characteristic decay time of the direct activation of oxidative phosphorylation. It is also shown that the size of PCr overshoot is increased by low resting PCr/Cr ratio (what is confirmed by our unpublished experimental data), by high intensity of the direct activation of oxidative phosphorylation, by high muscle work intensity and by low rate of the return of cytosolic pH to the resting value during muscle recovery.
Assuntos
Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Fosfocreatina/fisiologia , Difosfato de Adenosina/fisiologia , Trifosfato de Adenosina/fisiologia , Simulação por Computador , Creatina/fisiologia , Modelos Biológicos , Fosforilação Oxidativa , Esforço Físico/fisiologiaRESUMO
Although the exact mechanism(s) responsible for the phosphocreatine/ATP overshoot have not been completely elucidated, our data demonstrate that the overshoot does not stem from reduced myocardial work, and consequently, reduced utilization of phosphocreatine (PCr). Additionally, we highlight a basic difference in the physiologic responses of skeletal and cardial muscle to work demands. By understanding the bioenergetic derangements which accompany reperfusion injury, one may hope to better salvage post-ischemic myocardium.
Assuntos
Trifosfato de Adenosina/fisiologia , Coração/fisiologia , Fosfocreatina/fisiologia , Trifosfato de Adenosina/análise , Animais , Metabolismo Energético , Técnicas In Vitro , Masculino , Reperfusão Miocárdica , Miocárdio/química , Miocárdio/metabolismo , Consumo de Oxigênio , Fosfocreatina/química , Pressão , Ratos , Ratos Sprague-DawleyRESUMO
Towards understanding the role of glycolysis on synaptic function, we examined the effect of lactate on synaptic potential, energy metabolism, Ca(2+) homeostasis and extracellular glutamate in the dentate gyrus of guinea-pig hippocampus. Postsynaptic population spikes were recorded from the granule cell layer of the dentate gyrus in guinea-pig hippocampal slices after replacing glucose with lactate in the perfusion medium. Population spikes were not maintained and spontaneously recovered around 35min after the replacement of glucose with lactate. However, ATP levels of the dentate gyrus remained unchanged while those during the glucose-free condition decreased to 73% of the initial levels at 60min. Intracellular Ca(2+) was measured with the calcium indicator dye fura-2 AM, and the population spike was recorded simultaneously. Ca(2+) levels increased concomitantly with the early decay of synaptic potentials, and recovered partially with the spontaneous recovery of synaptic potentials. The time course of decay of population spikes and the increase of Ca(2+) levels during lactate replacement were similar to those during glucose deprivation. Increase in Ca(2+) levels during lactate replacement was completely blocked by the ryanodine receptor/calcium release channel antagonist dantrolene. Glutamate was released more significantly in the medium during lactate replacement than with normal Ringer solution, and less than that during glucose deprivation. Addition of the N-methyl-D-aspartate blocker, D-(-)-2-amino-5-phosphonovaleric acid, and the L-type calcium channel blocker, nimodipine, but not dantrolene blocked spontaneous recovery of population spikes. The results indicate that lactate can maintain energy levels in hippocampal slices, but cannot maintain ion homeostasis in granule cells of the dentate gyrus. Glycolysis plays an important role in maintaining ion homeostasis, and activation of N-methyl-D-aspartate and L-type calcium channels is necessary for support of synaptic function by lactate utilization.
Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Giro Denteado/metabolismo , Ácido Glutâmico/metabolismo , Glicólise/fisiologia , Ácido Láctico/metabolismo , Neurônios/metabolismo , 2-Amino-5-fosfonovalerato/farmacologia , Potenciais de Ação/efeitos dos fármacos , Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Giro Denteado/citologia , Giro Denteado/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Cobaias , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Ácido Láctico/farmacologia , Neurônios/efeitos dos fármacos , Nimodipina/farmacologia , Técnicas de Cultura de Órgãos , Fosfocreatina/efeitos dos fármacos , Fosfocreatina/fisiologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Teofilina/análogos & derivados , Teofilina/farmacologiaRESUMO
Prolactin (PRL)-secreting GH3 cells were grown, in vitro, with the creatine analogue beta-guanidinopropionic acid (GPA) added to the culture medium. After 5 days there was a small increase in basal and greatly increased thyrotropin-releasing hormone (TRH)-stimulated PRL secretion. The site of action of GPA is at the TRH-induced hydrolysis of phosphoinositides, since increased amounts of mono, bis and tris/tetrakis inositol phosphates were found in treated cells, while the PRL secretion induced by a phorbol ester or a calcium ionophore, treatments which mimic the second messages generated by inositol phospholipid hydrolysis, were not enhanced by GPA. The mechanism by which GPA increases phospholipase C activity has not been fully elucidated but may involve the activity of a controlling G protein.
Assuntos
Creatina/análogos & derivados , Guanidinas/farmacologia , Fosfatidilinositóis/metabolismo , Prolactina/metabolismo , Propionatos/farmacologia , Hormônio Liberador de Tireotropina/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Aminoquinolinas , Animais , Calcimicina/farmacologia , Relação Dose-Resposta a Droga , Hidrólise , Fosfatos de Inositol/biossíntese , Lactatos/biossíntese , Lítio/farmacologia , Fosfocreatina/fisiologia , Neoplasias Hipofisárias/tratamento farmacológico , Neoplasias Hipofisárias/metabolismo , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/metabolismoRESUMO
Intracellular kinetics of high energy phosphate (HEP) is of fundamental importance in cellular biochemical physiology. In mammalian brain, intracellular HEP transport from the production site (mitochondria) to the consumption site (plasma membrane) is dependent on passive diffusion of HEP through the cytosol. Diffusivity of a substrate in a solution correlates inversely to the viscosity of the solution. The maturational process of mammalian brain involves dramatic changes in the cytosolic amino acid profile. Since the viscosity of a solution is a function of the diffusion coefficients of solutes and their concentrations, changes in the cytosolic amino acid composition should result in significant alteration in cytosol viscosity and hence, HEP diffusivity. Such a system is especially suitable for mathematical modeling and correlative analysis by in vivo nuclear magnetic resonance (NMR) spectroscopy. This brief review is written to provide a fundamental background for investigational methodologies on developmental neurobiology of cellular energetics.
Assuntos
Encéfalo/fisiologia , Transferência de Energia/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Transporte Biológico/fisiologia , Citoplasma/fisiologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Fosfocreatina/fisiologia , RatosRESUMO
OBJECTIVE: To determine the effects of phosphocreatine (PCr) depletion on myocellular energetics. DESIGN: Randomized controlled study. SETTING: University laboratory. MATERIALS: Thirty-eight adult male Wistar rats (110-121 g). METHODS: The poorly metabolized creatine analogue beta-guanidinopropionic acid, (beta-GPA, 2% of a gel diet) was fed to the rats for 14 days to replace (75%) endogenous PCr stores before cecal ligation and puncture (CLP). Rats were randomized to receive sham operation and gel diet (sham-gel group [n=10]), sham operation and beta-GPA diet (sham-beta-GPA group [n=9]), CLP and gel diet (CLP-gel group [n=10]), and CLP and beta-GPA diet (CLP-beta-GPA group [n=9]). On day 14, all animals underwent operation. Twenty-four hours later, in vivo phosphorus 31-labeled magnetic resonance spectroscopy (31P-MRS) of the gastrocnemius muscle was performed. Muscle samples were collected to determine enzyme activities of beta-hydroxyacyl-CoA dehydrogenase, phosphofructokinase, citrate synthase, and the metabolites adenosine triphosphate (ATP), PCr, inorganic phosphate, and creatine. Free adenosine diphosphate levels, the phosphorylation potential, and free energy change of ATP hydrolysis were then calculated. RESULTS: All animals undergoing CLP but no controls had positive results of blood cultures. Although sham-beta-GPA animals had altered bioenergetics, CLP-beta-GPA rats experienced a greater deterioration of energy state compared with CLP-gel controls. Glycolytic and oxidative enzyme activities were not significantly different between groups and therefore could not explain the observed differences. CONCLUSIONS: There is an overall decrease in energy availability during sepsis, which is worsened by PCr depletion. These changes support the contention that PCr plays an important role as an ATP buffer during systemic infection.
Assuntos
Metabolismo Energético , Músculo Esquelético/metabolismo , Fosfocreatina/fisiologia , Sepse/metabolismo , Sepse/fisiopatologia , Animais , Masculino , Músculo Esquelético/citologia , Ratos , Ratos WistarRESUMO
The role of newly synthesized ATP in cyclic GMP-induced relaxation was studied in membrane permeabilized longitudinal muscle preparations of the rat proximal colon. Cyclic GMP and 8 bromo cGMP induced concentration-dependent relaxation of alpha-toxin permeabilized preparations which were precontracted by 3 microM Ca2+ in the presence of 4 mM ATP and 5 mM phosphocreatine (PC). The relaxation by 8 bromo cGMP was inhibited by Rp-8-pCPT cGMPS, an inhibitor of cyclic GMP dependent protein kinase. The relaxation was inhibited by removal of PC from the bathing solution, in spite of the presence of ATP. The relaxation was also inhibited by dinitrofluorobenzene (DNFB), a selective inhibitor of creatine kinase. The removal of PC or treatment with DNFB is known to produce accumulation of ADP within smooth muscle cell, however, ADPbetaS did not affect the relaxation. After irreversible inhibition of endogenous creatine kinase by DNFB in beta-escin permeabilized preparations, treatment of the preparations with exogenous creatine kinase restored the relaxation. In the presence of ADP and PC but without ATP, 8-bromo cGMP induced the relaxation to the similar extent to that in the presence of ATP and PC. These results suggest that ATP newly synthesized from ADP and PC by creatine kinase is essential for cyclic GMP-induced relaxation of the smooth muscle preparations obtained from the proximal colon of rats.
Assuntos
Trifosfato de Adenosina/fisiologia , Toxinas Bacterianas/farmacologia , Colo/efeitos dos fármacos , GMP Cíclico/farmacologia , Proteínas Hemolisinas/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Animais , Cálcio/fisiologia , Colo/fisiologia , Técnicas In Vitro , Masculino , Músculo Liso/fisiologia , Fosfocreatina/fisiologia , Ratos , Ratos WistarRESUMO
Cytoplasmic alpha-glycerol-3-phosphate dehydrogenase from fruit-bat-breast muscle was purified by ion-exchange and affinity chromatography. The specific activity of the purified enzyme was approximately 120 units/mg of protein. The apparent molecular weight of the native enzyme, as determined by gel filtration on Sephadex G-100 was 59,500 +/- 650 daltons; its subunit size was estimated to be 35,700 +/- 140 by SDS-polyacrylamide gel electrophoresis. The true Michaelis-Menten constants for all substrates at pH 7.5 were 3.9 +/- 0.7 mM, 0.65 +/- 0.05 mM, 0.26 +/- 0.06 mM, and 0.005 +/- 0.0004 mM for L-glycerol-3-phosphate, NAD(+), DHAP, and NADH, respectively. The true Michaelis-Menten constants at pH 10.0 were 2.30 +/- 0.21 mM and 0.20 +/- 0.01 mM for L-glycerol-3-phosphate and NAD(+), respectively. The turnover number, k(cat), of the forward reaction was 1.9 +/- 0.2 x 10(4)s(-1). The treatment of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) under denaturing conditions indicated that there were a total of eight cysteine residues, while only two of these residues were reactive towards DTNB in the native enzyme. The overall results of the in vitro experiments suggest that alpha-glycerol-3-phosphate dehydrogenase of the fruit bat preferentially catalyses the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate.
Assuntos
Quirópteros , Glicerolfosfato Desidrogenase/metabolismo , Músculos Peitorais/enzimologia , Trifosfato de Adenosina/fisiologia , Aminoácidos/análise , Animais , Citoplasma/enzimologia , Fosfato de Di-Hidroxiacetona/metabolismo , Ácido Ditionitrobenzoico/metabolismo , Inibidores Enzimáticos/farmacologia , Glicerol-3-Fosfato Desidrogenase (NAD+) , Glicerolfosfato Desidrogenase/química , Glicerolfosfato Desidrogenase/isolamento & purificação , Glicerofosfatos/farmacologia , Cinética , Peso Molecular , Fosfocreatina/fisiologiaRESUMO
The principal motivation for the present work was the study of the kinetics of anaerobic metabolism. A new mathematical model of the bioenergetics of sprinting, incorporating a three-equation representation of anaerobic metabolism, is developed. Results computed using the model are compared with measured data from the mens' finals of the 100m event at the 1987 World Championships. The computed results closely predict the overall average performance of the competitors over the course of the entire race. Further calculations show the three-equation model of anaerobic metabolism to be a significant improvement over the previous one-equation model. Representative values of time constants that govern the rate of anaerobic energy release have been determined for elite male athletes. For phosphocreatine utilisation, values for lambda(2)=0. 20s(-1) and psi(2)=3.0s(-1) are consistent with data previously reported in the literature. New values of lambda(3)=0.033s(-1) and psi(3)=0.34s(-1) are proposed as offering an improved representation of the kinetics of oxygen-independent glycolysis. For the first time, tentative values for the time constants of ATP utilisation, lambda(1)=0.9s(-1) and psi(1)=20s(-1), are suggested. The maximum powers developed during sprinting by oxygen-independent glycolysis, PCr utilisation and endogenous ATP utilisation were calculated as 34. 1, 30.1 and 16.6Wkg(-1), respectively, with an overall maximum anaerobic power of 51.6Wkg(-1). Sample calculations show the mathematical model can be used in principle to derive data on the kinetics of anaerobic metabolism of individual athletes.
Assuntos
Metabolismo Energético/fisiologia , Corrida/fisiologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/fisiologia , Glicólise/fisiologia , Humanos , Cinética , Masculino , Modelos Biológicos , Movimento , Fosfocreatina/metabolismo , Fosfocreatina/fisiologia , Aptidão Física , Sensibilidade e Especificidade , Fatores de TempoRESUMO
In this paper we develop a theory for calculating the chemical energy liberation and heat production of a skeletal muscle subjected to an arbitrary history of stimulation, loading, and length variation. This theory is based on and complements the distribution-moment (DM) model of muscle [Zahalak and Ma, J. biomech. Engng 112, 52-62 (1990)]. The DM model is a mathematical approximation of the A. F. Huxley cross-bridge theory and represents a muscle in terms of five (normalized) state variables: A, the muscle length, c, the sarcoplasmic free calcium concentration, and Q0, Q1, Q2, the first three moments of the actin-myosin bond-distribution function (which, respectively, have macroscopic interpretations as the muscle stiffness, force, and elastic energy stored in the contractile tissue). From this model are derived two equations which predict the chemical energy liberation and heat production rates in terms of the five DM state variables, and which take account of the following factors: (1) phosphocreatine hydrolysis associated with cross-bridge cycling; (2) phosphocreatine hydrolysis associated with sarcoplasmic-reticulum pumping of calcium; (3) passive calcium flux across the sarcoplasmic-reticulum membrane; (4) calcium-troponin bonding; (5) cross-bridge bonding at zero strain; (6) cross-bridge strain energy; (7) tendon strain energy; and (8) external work. Using estimated parameters appropriate for a frog sartorius at 0 degree C, the energy rates are calculated for several experiments reported in the literature, and reasonable agreement is found between our model and the measurements. (The selected experiments are confined to the plateau of the isometric length-tension curve, although our theory admits arbitrary length variations.) The two most important contributions to the energy rates are phosphocreatine hydrolysis associated with cross-bridge cycling and with sarcoplasmic-reticulum calcium pumping, and these two contributions are approximately equal under tetanic, isometric, steady-state conditions. The contribution of the calcium flux across the electrochemical potential gradient at the sarcoplasmic-reticulum membrane was found to be small under all conditions examined, and can be neglected. Long-term fatigue and oxidative recovery effects are not included in this theory. Also not included is the so-called 'unexplained energy' presumably associated with reactions which have not yet been identified. Within these limitations our model defines clear quantitative interrelations between the activation, mechanics, and energetics in muscle, and permits rational estimates of the energy production to be calculated for arbitrary programs of muscular work.
Assuntos
Metabolismo Energético , Modelos Biológicos , Contração Muscular/fisiologia , Músculos/fisiologia , Actinas/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Anuros , Cálcio/fisiologia , Elasticidade , Metabolismo Energético/fisiologia , Temperatura Alta , Humanos , Músculos/metabolismo , Miosinas/fisiologia , Fosfocreatina/fisiologia , Retículo Sarcoplasmático/fisiologia , Estresse Mecânico , Tendões/fisiologia , Termodinâmica , Troponina/fisiologiaRESUMO
The present study was designed to elucidate metabolic factors related to reoxygenation-induced recovery of cardiac contractile force after a period of hypoxia, from the view point of energy metabolism in the myocardium. Rabbit hearts were perfused for 20 mins under various degrees of hypoxic conditions, followed by 45 mins of reoxygenation. Hypoxia induced a rise in resting tension, a cessation of cardiac contractile force, a depletion of high energy phosphates, an increase in tissue calcium and an increase in UV absorbance of the perfusate. High performance liquid chromatography analysis of the perfusate indicated that the increase in UV absorbance of the perfusate was attributed to the release of ATP metabolites from the perfused heart. Reoxygenation-induced recovery of cardiac contractile force after 20 mins of hypoxia was predicted by the degree of the rise in resting tension at the final period of hypoxia. The recovery was related to the level of high energy phosphates in the reoxygenated heart as well as the loss of ATP metabolites from the heart but not to the tissue calcium content. The loss of ATP metabolites also correlated with myocardial ATP levels at 45 mins of reoxygenation and a rise in resting tension at 20 mins of hypoxia. The results suggest that loss of ATP metabolites is a vital step in the induction of incomplete recovery of cardiac contractile force after hypoxia.
Assuntos
Trifosfato de Adenosina/fisiologia , Contração Miocárdica , Miocárdio/metabolismo , Oxigênio/fisiologia , Fosfocreatina/fisiologia , Animais , Cálcio/metabolismo , Cromatografia Líquida de Alta Pressão , Metabolismo Energético , Feminino , Coração , Técnicas In Vitro , Masculino , Consumo de Oxigênio , Perfusão , CoelhosRESUMO
A general understanding of the pivotal role of phosphocreatine (PCr) as the principal determinant of skin flap survival is now emerging. Definitive metabolic investigations using phosphorus (31P) and proton (1H) magnetic resonance spectroscopy (MRS) have established that the inability to replenish metabolically exhausted PCr reserves predictably correlates with skin flap necrosis. Furthermore, postoperative parenteral administration of PCr has been shown to augment effectively skin flap survival. We hypothesized that creatine kinase, the enzyme controlling the utilization of the high-energy phosphate component of PCr, is a critical determinant of the tolerance of a skin flap to ischemic insult. In other words, if the rate of utilization of PCr is too rapid, PCr stores will rapidly deplete, and the flap will not be able to withstand a period of ischemia. Alternatively, if the rate of dephosphorylation of PCr is reduced, survival of skin flaps during periods of ischemia could be extended. To test this hypothesis, we investigated the metabolic distribution and fate of cyclocreatine (cCr), a competent creatine analogue with a lower affinity for the creatine kinase enzyme. When administered as 1.5 percent (w/w) of the normal diet of laboratory rats, cCr accumulates in skin as the competent phosphagen, phosphocyclocreatine (PcCr). Cutaneous flaps elevated in these animals, and studied by 31P and 1H MRS, demonstrate that once depletion of PCr has occurred, PcCr continues to sustain ATP levels. This results in significant enhancement of skin flap survival (p < 0.005). These observations confirm the importance of the creatine kinase enzyme in cutaneous flap ischemia and suggest new approaches to augment skin flap survival.
Assuntos
Creatina Quinase/fisiologia , Metabolismo Energético/fisiologia , Imidazolidinas , Fosfocreatina/fisiologia , Traumatismo por Reperfusão/prevenção & controle , Retalhos Cirúrgicos/irrigação sanguínea , Trifosfato de Adenosina/metabolismo , Animais , Creatinina/administração & dosagem , Creatinina/análogos & derivados , Creatinina/farmacocinética , Sobrevivência de Enxerto/fisiologia , Masculino , Fosfocreatina/análogos & derivados , Fosfocreatina/metabolismo , Ratos , Traumatismo por Reperfusão/metabolismo , Retalhos Cirúrgicos/patologiaRESUMO
Human lymphoblastoid Raji cells and mouse hybridoma ascites cells incubated with 20 mM creatine showed significant increases in creatine, creatine phosphate and adenine nucleotide levels and in the energy charge. In human erythrocytes in which no variation of the creatine phosphate-creatine pool was observed because of a very low creatine kinase activity, the adenine nucleotide pool and the energy charge were not modified. These observations suggest not only a relationship among the creatine phosphate-creatine pool, the energy charge and the adenylate pool, but also the possibility to increase the energy charge and the adenylate pool in cells with creatine kinase activity by expanding the creatine phosphate-creatine pool.