RESUMEN
Mammalian brain is a highly oxidative organ and although it constitutes only a small fraction of total body weight it accounts for a disproportionately large percentage of bodily oxygen consumption (in humans about 2 and 20%, respectively). Yet, the partial pressure and concentration of oxygen in the brain are low and non-uniform. There is a large number of enzymes that use O(2) as a substrate, the most important of which is cytochrome c oxidase, the key to mitochondrial ATP production. The affinity of cytochrome c oxidase for oxygen is very high, which under normal conditions ensures undiminished activity of oxidative phosphorylation down to very low P(O(2)). By contrast, many other relevant enzymes have K(m) values for oxygen within, or above, the ambient cerebral gas tension, thus making their operations very dependent on oxygen level in the physiological range. Among its multiple, versatile functions, oxygen partial pressure and concentration control production of reactive oxygen species, expression of genes and functions of ion channels. Limitation of oxygen supply to the brain below a 'critical' level reduces, and eventually blocks oxidative phosphorylation, drastically decreases cellular (ATP) and leads to a collapse of ion gradients. Neuronal activity ceases and if oxygen is not re-introduced quickly, cells die. The object of this review is to discuss briefly the central oxygen-dependent processes in mammalian brain and the short-term consequences of O(2) deprivation, but not the mechanisms of long-term adaptation to chronic hypoxia. Particular emphasis is placed on issues which have been the focus of recent attention and/or controversy.
Asunto(s)
Encéfalo/fisiología , Hipoxia Encefálica/fisiopatología , Consumo de Oxígeno/fisiología , Animales , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In a cell culture model of murine osteoblasts three particulate bioactive glasses were evaluated and compared to glass (either borosilicate or soda-lime-silica) particles with respect to their effect on metabolic activity, cell viability, changes in intracellular ion concentrations, proliferation and differentiation. 45S5 Bioglass caused extra- and intracellular alkalinization, a rise in [Ca2+]i and [K+]i, a small plasma membrane hyperpolarization, and an increase in lactate production. Glycolytic activity was also stimulated when cells were not in direct contact with 45S5 Bioglass particles but communicated with them only through the medium. Similarly, raising the pH of culture medium enhanced lactate synthesis. 45S5 Bioglass had no effect on osteoblast viability and, under most conditions, did not affect either proliferation or differentiation. Bioactive glasses 58S and 77S altered neither the ion levels nor enhanced metabolic activity. It is concluded that: (1) some bioactive glasses exhibit well-defined effects in osteoblasts in culture which are accessible to experimentation; (2) 45S5 Bioglass causes marked external and internal alkalinization which is, most likely, responsible for enhanced glycolysis and, hence, cellular ATP production; (3) changes in [H+] could contribute to alternations in concentrations of other intracellular ions; and (4) the rise in [Ca2+]i may influence activities of a number of intracellular enzymes and pathways. It is postulated that the beneficial effect of 45S5 on in vivo bone growth and repair may be due to some extent to alkalinization, which in turn increases collagen synthesis and crosslinking, and hydroxyapatite formation.
Asunto(s)
Materiales Biocompatibles/farmacología , Cerámica/farmacología , Osteoblastos/citología , Osteoblastos/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Osteoblastos/efectos de los fármacos , Cráneo , Relación Estructura-ActividadRESUMEN
The present study characterized metabolic changes in the heart associated with long-term exposure to hypoxia, a potent stimulus for pulmonary hypertension and right ventricular hypertrophy. When anesthetized rats adapted to chronic hypoxia spontaneously respired room air, their mean right intraventricular peak systolic pressure (RVSP) was twice that in normal control animals with the same arterial PO2. RVSP was linearly related to right ventricular mass (r = 0.78). Oxidative capacity (O2 consumption) of homogenates of right and left ventricles from both groups of rats was measured with one of the following substrates: pyruvate, glutamate, acetate, and palmitoyl-L-carnitine. Oxidation of all substrates was significantly greater in the left than in the right ventricle in normal rats but not in hypoxia-adapted animals, where it was the same, within the experimental error. O2 consumption by the left ventricle was greater in control than in experimental rats, but right ventricular O2 consumption was similar in the two groups. Maximal reaction velocity of cytochrome-c oxidase was about the same in the two ventricles, and there were no significant differences between control and hypoxia-adapted animals. HPLC analyses showed significantly higher aspartate levels and aspartate-to glutamate concentration ratios in both ventricles of hypoxic rats than in corresponding tissues from controls, indicative of a decreased flux through the malate-aspartate shuttle under conditions of O2 limitation. Myocardial glutamine levels were lower in hypoxic rats, and glutamine-to-glutamate concentration ratios decreased, although primarily in the pressure-overloaded right ventricle. These findings indicate that normal energy metabolism in the left ventricle differs from that in the right and that the differences, particularly those of amino acid metabolism, are markedly influenced by chronic exposure to hypoxia.
Asunto(s)
Adaptación Fisiológica/fisiología , Metabolismo Energético/fisiología , Hipoxia/fisiopatología , Miocardio/metabolismo , Aminoácidos/metabolismo , Animales , Presión Sanguínea/fisiología , Glucólisis/fisiología , Ventrículos Cardíacos , Hipoxia/metabolismo , Masculino , Oxidación-Reducción , Consumo de Oxígeno/fisiología , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Sístole , Función Ventricular Derecha/fisiologíaRESUMEN
Several inhibitors of mitochondrial complex II cause neuronal death in vivo and in vitro. The goal of the present work was to characterize in vitro the effects of malonate (a competitive blocker of the complex) which induces neuronal death in a pattern similar to that seen in striatum in Huntington's disease. Exposure of striatal and cortical cultures from embryonic rat brain for 24 h to methylmalonate, a compound which produces malonate intracellularly, led to a dose-dependent cell death. Methylmalonate (10 mM) caused >90% mortality of neurons although cortical cells were unexpectedly more vulnerable. Cell death was attenuated in a medium containing antioxidants. Further characterization revealed that DNA laddering could be detected after 3 h of treatment. Morphological observations (videomicroscopy and Hoechst staining) showed that both necrotic and apoptotic cell death occurred in parallel; apoptosis was more prevalent. A decrease in the ATP/ADP ratio was observed after 3 h of treatment with 10 mM methylmalonate. In striatal cultures it occurred concomitantly with a decline in GABA and a rise in aspartate content and the aspartate/glutamate ratio. Changes in ion concentrations were measured in similar cortical cultures from mouse brain. Neuronal [Na+]i increased while [K+]i and membrane potential decreased after 20 min of continuous incubation in 10 mM methylmalonate. These changes progressed with time, and a rise in [Ca2+]i was also observed after 1 h. The results demonstrate that malonate collapses cellular ion gradients, restoration of which imposes an additional load on the already compromised ATP-generation machinery. An early elevation in [Ca2+]i may trigger an increase in activity of proteases, lipases and endonucleases and production of free radicals and DNA damage which, ultimately, leads to cells death. The data also suggest that maturational and/or extrinsic factors are likely to be critical for the increased vulnerability of striatal neurons to mitochondrial inhibition in vivo.
Asunto(s)
Apoptosis , Encéfalo/citología , Corteza Cerebral/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Ácido Metilmalónico/toxicidad , Neuronas/efectos de los fármacos , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Ácido Aspártico/metabolismo , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/metabolismo , Cuerpo Estriado/metabolismo , Complejo II de Transporte de Electrones , Feto , Ácido Glutámico/metabolismo , Cinética , Ratones , Microscopía por Video , Complejos Multienzimáticos/antagonistas & inhibidores , Neuronas/metabolismo , Neuronas/patología , Oxidorreductasas/antagonistas & inhibidores , Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Sodio/metabolismo , Succinato Deshidrogenasa/antagonistas & inhibidores , Factores de Tiempo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
In the lateral hypothalamic area (LHA) of rat brain, approximately 30% of cells showed sensitivity to small changes in local concentrations of glucose. These "glucose-sensitive" neurons demonstrated four types of behavior, three of which probably represent segments of a continuous spectrum of recruitment in response to ever more severe changes in blood sugar. Type I cells showed maximum activity =5.6 mM blood glucose but became completely silent at hyperglycemia of 10-12 mM (normoglycemia 7.6 +/- 0.3 mM; mean +/- SD). Type II and III neurons exhibited a wider range of response. Type IV cells (5-7% of glucose-sensitive neurons) paralleled the behavior of sugar-sensitive cells in ventromedial hypothalamic nucleus (VMH). In VMH, approximately 40% of cells responded to changes in blood glucose over a range of concentrations from 3.6 to 17 mM, by increasing their firing rate as sugar level rose and vice versa. Ionic shifts during increases in blood (brain) glucose levels were similar in LHA types I-III but fastest in I and slowest in III. [Na+]i fell by 5-9 mM, [K+]i rose by 6-8 mM, and plasma membrane hyperpolarized by 5 mV. [Ca2+]i declined by 15-20 nM in line with membrane hyperpolarization. In VMH and type IV LHA cells, [K+]i fell 3-8 mM and plasma membrane depolarized -3 to -5 mV as blood/brain glucose concentration increased from 7.6/2.4 to 17.6/4.2 mM, whereas [Ca2+]i increased from 125 to 180 nM as a consequence of falling membrane potential. During falls in blood/brain sugar concentration the effects in both VMH and LHA cells were reversed. The findings are consistent with the ionic shifts in types I-III LHA cells being dependent on alterations in Na/K-ATPase activity, whereas those in VMH and type IV LHA cells could be caused by modulation of ATP-dependent K+ channels. A possible mechanism for linking the effects of small changes in glucose to ATP generation, which could bring about the above phenomena, is the interposition of a "glucokinase-type" enzyme in a role similar to that which it has in glucose-sensing pancreatic beta-cells.
Asunto(s)
Glucemia/metabolismo , Cationes/metabolismo , Área Hipotalámica Lateral/metabolismo , Hipotálamo Medio/metabolismo , Neuronas/metabolismo , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Femenino , Área Hipotalámica Lateral/citología , Hipotálamo Medio/citología , Masculino , Potenciales de la Membrana/fisiología , Microelectrodos , Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Sodio/metabolismoRESUMEN
Intrastriatal injections of the mitochondrial toxins malonate and 3-nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntington's disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose-dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 microM) nor dopamine (2.5 microM) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 microM) did not alter the ATP/ADP ratio after a 6-h incubation. Methyl malonate, at 500 microM, reduced the ATP/ADP ratio by approximately 30% after 6 h; this decrease was not augmented by coincubation with 25 microM dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Dopamina/toxicidad , Malonatos/toxicidad , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Adenosina Trifosfato/antagonistas & inhibidores , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Benzazepinas/farmacología , Células Cultivadas , Cuerpo Estriado/metabolismo , Cuerpo Estriado/ultraestructura , ADN/análisis , Dopamina/metabolismo , Antagonistas de Dopamina/farmacología , Inhibidores de Captación de Dopamina/farmacología , Sinergismo Farmacológico , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Depuradores de Radicales Libres/farmacología , Mitocondrias/metabolismo , Neuronas/metabolismo , Neuronas/ultraestructura , Nomifensina/farmacología , Norepinefrina/farmacología , Ratas , Ratas Sprague-Dawley , Coloración y Etiquetado , Factores de Tiempo , Grabación en VideoRESUMEN
Changes in neuronal activity and extracellular concentrations of ions were measured in rat striatum for 60-90 min after intrastriatal injection of quinolinic acid, an agonist of the N-methyl-D-aspartate receptor. The excitotoxin induced bursts of synchronous electrical activity which were accompanied by rises in [K+]e (to approximately 6 mM) and decreases in [Ca2+]e (by less than 0.1 mM); [H+]e usually increased (0.1-0.3 pH unit) after a short and small (< 0.1 pH unit) alkaline shift. The magnitude and frequency of these periodic changes decreased with time; after 90 min the amplitudes fell to 10-20% of the early values and the frequency to about one every 8 min as compared to one every 2-3 min immediately after quinolinate injection. By 90 min there was an increase in [K+]e from 3.3 mM to 4.2 mM and a decrease in [Ca2+]e from 1.34 mM to 1.30 mM. It is postulated that activation of the N-methyl-D-aspartate receptor causes disturbances in neuronal activity and ion gradients; restoration of the original ionic balances raises utilization of ATP and places an additional demand on energy-producing pathways. Increased influx of calcium into neurons may lead to an enhanced accumulation and subsequent overload of mitochondria with the cation. This, in turn, could result in dysfunction of the organelles and account for the decrease in respiration and [ATP]/[ADP] that have been observed previously in this model. The results of the present study lead to the conclusion that quinolinic acid produces early changes in activity of striatal neurons and movements of several cations which may contribute to subsequent abnormalities in energy metabolism and ultimately, cell death.
Asunto(s)
Neostriado/metabolismo , Ácido Quinolínico/farmacología , Animales , Calcio/metabolismo , Electrofisiología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Hidrógeno/metabolismo , Masculino , Microelectrodos , Neostriado/citología , Neostriado/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Potasio/metabolismo , Ácido Quinolínico/administración & dosificación , Ratas , Ratas Sprague-DawleyRESUMEN
The goal of the present contribution is to discuss the relationships among brain oxygen tension, energy (ATP) level, and ion gradients and movements. The function of the CNS, the generation and transmission of impulses, is determined to a large extent by the movements of ions. Hence elucidation of these relationships is necessary to the understanding of how brain works. Moreover, such knowledge is indispensable for the design of rational therapies for treatment of a large group of pathological states caused by lack of oxygen. This paper is partly a review and partly an original contribution although the former involves to a considerable extent, results obtained in our laboratories. It is divided into 3 parts: a) a very brief general introduction which reminds the reader some well-known facts; b) presentation and discussion of data; and c) conclusions and/or predictions.
Asunto(s)
Encéfalo/metabolismo , Hipoxia de la Célula , Hipoxia Encefálica/metabolismo , Consumo de Oxígeno , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Humanos , Iones , Neuroglía/metabolismo , Neuronas/metabolismoRESUMEN
Impairment of mitochondrial energy metabolism may contribute to the selective neuronal degeneration observed in Huntington's disease and other neurodegenerative disorders. Intrastriatal injection of the excitotoxin, quinolinic acid, produces a pattern of neuronal death similar to that seen in Huntington's disease. However, little is known about the effects of quinolinic acid on striatal energetics. In the present work, time-dependent changes in energy metabolism caused by injection of quinolinic acid into rat striatum were examined. Oxygen consumption by free and synaptic mitochondria was quantified and correlated with the concentrations of nucleotides and amino acids at different times after injection. Compared with saline-treated controls, a decrease in ADP-stimulated (state 3) to basal (state 4) oxygen consumption (respiratory control ratio) by free mitochondria was apparent in quinolinic acid-injected striata as early as 6 h after treatment. No significant changes were seen in nucleotide concentrations at this time. By 12 h after injection, the decline in the respiratory control ratio was more pronounced (45%), and reductions in ATP, NAD, aspartate, and glutamate (30-60%) were also observed. These results show that injection of quinolinic acid in vivo produces progressive mitochondrial dysfunction, which may be a common and critical event in the cell death cascade initiated in Huntington's disease and in animal models of this neurodegenerative disorder. The indicators of mitochondrial function examined in this study, therefore, may be useful in evaluating the efficacy of neuroprotective agents.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Metabolismo Energético , Ácido Quinolínico/farmacología , Aminoácidos/metabolismo , Animales , Cuerpo Estriado/patología , Masculino , Mitocondrias/metabolismo , Nucleótidos/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Cultured astrocytes and cell lines derived therefrom maintain a high energy level ([ATP]/[ADP]) through operation of oxidative phosphorylation and glycolysis. The contribution from the latter to total ATP production is 25-32%. A powerful Na+/K+ pump maintains potassium, sodium, and calcium gradients out of equilibrium. [Na+]i is about 20 mM, [K+]i is 130 mM and [Ca2+]i is less than 100 nM. Under non-stimulated conditions, the Na+/K+ ATPase consumes 20% of astrocytic ATP production. Inhibition of the pump by ouabain decreases energy expenditure, raises [creatine phosphate]/[creatine], and leads to a leakage of sodium, potassium, and calcium ions. Decrease in the pump function via a fall in [ATP] also collapses ion gradients; the rate and extent of the fall correlates positively with cellular energy state. Under "normal" conditions (i.e., when ATP production pathways are not inhibited), there appears to be no preferential utilization of energy produced by either glycolysis or oxidative phosphorylation for the support of pump function.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Metabolismo Energético , Neuroglía/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Feto , Glioma , Concentración de Iones de Hidrógeno , Mamíferos , Ratones , Potasio/metabolismo , Sodio/metabolismo , Células Tumorales CultivadasRESUMEN
Intracellular concentrations of sodium, potassium and calcium together with membrane potentials were measured in cultured murine cortical neurons and glial cells under conditions which mimicked in vivo hypoxia, ischemia and hypoglycemia. These included; glucose omission with and without added pyruvate, addition of rotenone in the presence and absence of glucose and substitution of 2-deoxyglucose for glucose with and without rotenone. Cellular energy levels ([ATP], [ADP], [phosphocreatine], [creatine]) were measured in suspensions of C6 cells incubated in parallel under identical conditions. [Na+]i and [Ca2+]i rose while [K+]i fell and plasma membrane depolarized when energy production was limited. Intracellular acidification was observed when glycolysis was the sole source for ATP synthesis. There was a positive correlation between the extent of energy depletion in glial cells and the magnitude and velocity of alterations in ion levels. Neither glycolysis alone nor oxidative phosphorylation alone were able to ensure unaltered ion gradients. Since oxidative phosphorylation is much more efficient in generating ATP than glycolysis, this finding suggests a specific requirement of the Na pump for ATP generated by glycolysis. Changes in [Na+]i and [K+]i observed during energy depletion were gradual and progressive whereas those in [Ca2+]i were initially slow and moderate with large elevations occurring only as a late event. Increases in [Na+]i were usually smaller than reductions in [K+]i, particularly in the glia, suggestive of cellular swelling. Glia were less sensitive to identical insults than were neurons under all conditions. Results presented in this study lead to the conclusion that the response to energy deprivation of the two main types of brain cells, neurons and astrocytes, is a complex function of their capacity to produce ATP and the activities of various pathways which are involved in ion homeostasis.
Asunto(s)
Química Encefálica/fisiología , Encéfalo/citología , Metabolismo Energético/fisiología , Homeostasis/fisiología , Neuroglía/metabolismo , Neuronas/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Astrocitos/metabolismo , Células Cultivadas , Medios de Cultivo , Femenino , Glioma/metabolismo , Inmunohistoquímica , Potenciales de la Membrana/fisiología , Ratones , Nucleótidos/metabolismo , Fosforilación Oxidativa , Células Tumorales CultivadasRESUMEN
Stable isotopes were used to measure both the rate of GABA formation by glutamic acid decarboxylase (GAD) and the rate of utilization by GABA-transaminase (GABA-T). The initial rate of GABA accumulation, determined with either [2-15N]glutamine or [2H5]glutamine as precursor, was 0.3-0.4 nmol/min/mg of protein. Addition of the calcium ionophore A23187 enhanced GAD activity, whereas changes in levels of inorganic phosphate and H+ were without influence. Flux through GABA-T (GABA--> glutamate), measured with [15N]GABA as precursor, was 0.82 nmol/min/mg of protein, whereas the reamination of succinic acid semialdehyde (reverse flux through GABA-T) was almost sixfold faster, 4.8 nmol/min/mg of protein. The rate of GABA metabolism via the tricarboxylic acid cycle was very slow, with the upper limit on flux being 0.03 nmol/min/mg of protein. Addition of either acetoacetate or beta-hydroxybutyrate raised the internal content of glutamate and reduced that of aspartate; the GABA concentration and the rate of its formation increased. It is concluded that in synaptosomes (a) GABA-T is a primary factor in regulating the turnover of GABA, (b) a major regulator of GAD activity is the concentration of internal calcium, (c) GAD in nerve endings may not be saturated with its substrate, glutamate, and the concentration of the latter is a determinant of flux through this pathway, and (d) levels of ketone bodies increase, and maintain at a higher value, the synaptosomal content of GABA, a phenomenon that may contribute to the beneficial effect of a ketogenic diet in the treatment of epilepsy.
Asunto(s)
Calcio/farmacología , Ácido Glutámico/farmacología , Cuerpos Cetónicos/farmacología , Sinaptosomas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , 4-Aminobutirato Transaminasa/metabolismo , Animales , Encéfalo/enzimología , Glutamina/metabolismo , Masculino , Fosfatos/farmacología , Protones , Ratas , Ratas Sprague-Dawley , Sinaptosomas/efectos de los fármacos , Ácido gamma-Aminobutírico/biosíntesisRESUMEN
Incubation of rat brain synaptosomes under conditions of either increased energy utilization (addition of Na+ channel opener, veratridine, or ionophores, monensin and nigericin) or inhibition of oxidative phosphorylation (addition of rotenone), or a combination thereof, decreased [ATP], increased [ADP] and stimulated glycolysis. The rates of lactate generation were linear over a 15-min interval in the presence of rotenone alone but decreased in the other two conditions. During the first 5 min, the amount of lactate formed with veratridine, monensin or nigericin was as high or higher than with rotenone, but it was lower in the last 10 min. With a combination of one of the stimulators of ion movements and rotenone the rate of glycolysis was always markedly lower than with each compound added singly. The stimulated rates of lactate formation correlated positively with the synaptosomal content of [ATP]. After 15 min, [ATP] was 0.9-1.0 nmol/mg with rotenone, 0.5-0.9 nmol/mg with veratridine (or ionophores), and <0.3 nmol/mg with a combination of the two. Under the conditions used, calcium did not affect glycolytic activity directly. The Lineweaver-Burk plot of the rate of lactate formation against [ATP] yielded a straight line with a Km for ATP of about 0.1 mM, which is very similar to the Km for this nucleotide of brain hexokinase bound to mitochondria. In C6 cells glycolytic rate measured with a combination of an ionophore and rotenone was higher than with each of these compounds added singly while [ATP] never declined below about 9 nmol/mg prot. It is concluded that in synaptosomes, the high rate of energy utilization required for intense ion movement decreases [ATP] to a level that limits hexokinase activity kinetically. This may contribute to a reduction in the rate of glycolysis and hence energy production in brain hypoxia and ischemia.
Asunto(s)
Encéfalo/efectos de los fármacos , Glucólisis/efectos de los fármacos , Hexoquinasa/metabolismo , Bombas Iónicas/efectos de los fármacos , Sinaptosomas/efectos de los fármacos , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/ultraestructura , Ionóforos/farmacología , Masculino , Monensina/farmacología , Nigericina/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Sinaptosomas/metabolismo , Células Tumorales Cultivadas , Veratridina/farmacologíaRESUMEN
The metabolism of branched-chain amino acids (BCAAs) was studied in cortical synaptosomes. With [15N]leucine (1 mM) as precursor, the cumulative appearance of 15N in [15N]glutamate and [15N]aspartate was 0.2 nmol/min/mg of protein without supplemental alpha-ketoglutarate and 0.3 nmol/min/mg of protein in the presence of alpha-ketoglutarate (0.5 mM). The BCAA amino-transferase reaction also proceeded in the "reverse" direction [alpha-ketoisocaproate (KIC) + glutamate-->leucine + alpha-ketoglutarate]. This was documented by incubating synaptosomes with [15N]glutamate and measuring the formation of [15N]leucine. Without KIC in the medium, the rate of [15N]leucine production was 0.13 nmol/min/mg of protein. In the presence of 25 microM KIC the rate was 0.79 nmol/min/mg of protein and even greater (1.0 nmol/ min/mg of protein) in the presence of 500 microM KIC. The reamination of KIC was two- to threefold faster with [2-15N]glutamine as precursor compared with [15N]-glutamate. The ketoacid of valine, alpha-ketoisovalerate (KIV), was reaminated to [15N]valine at a rate comparable to that observed with respect to KIC. The BCAA transaminase mediated not only the bidrectional transfer of amino groups between leucine or valine and glutamate, but also the direct transfer of nitrogen between leucine and valine. This was ascertained in studies in which the incubation medium was supplemented with either [15N]leucine and KIV or [15N]valine and KIC (amino acids at 1 mM and ketoacids at 25 or 500 microM). The rate was faster in the direction of leucine formation at both the lower (6.1-fold) and higher (1.7-fold) KIC concentration. It is suggested that in synaptosomes the BCAA transaminase (a) functions predominantly in the direction of leucine formation and (b) maintains a constant ratio of BCAAs and ketoacids to one other.
Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Neuronas/metabolismo , Sinaptosomas/metabolismo , Transaminasas/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Cromatografía de Gases y Espectrometría de Masas , Nitrógeno/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Despite the complexity of the mechanisms that control free calcium concentration in neural cells, considerable advances have been made recently in the understanding of the entry and exit pathways of the ion through application of selective channel and receptor blockers. In addition, useful knowledge has been gained of the internal regulation of calcium movements and the factors that lead to mobilization of the ions into, or their sequestration from, the cytosol. It is clear that calcium homeostasis is crucial to cell metabolism and survival, and that relatively small deviations from the norm can have serious or lethal consequences. It appears that many of the experimental tools are now available to assist in the elucidation of the mechanisms controlling intracellular calcium concentration in vitro. Nevertheless, it has to be accepted that however valuable results from model systems may be, the final testing of any hypothesis concerning the importance of calcium homeostasis in the intact brain requires extensive experimental and clinical investigations in vivo. Information obtained in vitro, if convincingly confirmed by in vivo studies, may well be crucial in formulating strategies to combat a wide range of pathologic conditions.
Asunto(s)
Calcio/metabolismo , Hipocampo/metabolismo , Isquemia/metabolismo , AnimalesRESUMEN
The effect of [H+] on the rate of glycolysis was investigated in glioma C6 and fibroblast BHK-21 cells and in synaptosomes from rat brain. The rates of lactate production at an extracellular pH (pHe) of 6.2, 7.4, and 7.8 were correlated with intracellular [ATP], [ADP], and [P(i)] ([ATP]i, [ADP]i, and [P(i)]i, respectively) and, when relevant, creatine phosphate (PCr) as well as with the levels of several glycolytic intermediates. In C6 cells cytosolic [H+] was measured simultaneously together with [Ca2+], [K+], [Na+], and membrane potentials. In all three systems studied, an increase in [H+]e suppressed whereas a fall enhanced the rate of lactate generation. Changes in pHe produced no simple correlation between the amount of lactate formed and alterations either in the absolute [ATP], [ADP], [P(i)], and [PCr] or their ratios but did correlate with the levels of glycolytic intermediates. Higher [fructose-1,6-bisphosphate] and [glyceraldehyde-3-phosphate] and lower [glucose-6-phosphate] and [fructose-6-phosphate] accompanied faster glycolytic activity. Addition of rotenone markedly enhanced glycolysis at all pHe values studied. The increases were larger at higher [H+] so that the rate of lactate generation was only slightly lower at pH 6.2 than at 7.4 or 7.8. With rotenone present, [ATP] (and where relevant [PCr]) fell and [ADP] and [P(i)] rose under all pHe conditions. Simultaneously [glucose-6-phosphate] and [fructose-6-phosphate] decreased whereas [fructose-1,6-bisphosphate] and [glyceraldehyde-3-phosphate] increased; the levels of the last two were similar at pH 6.2 and 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Glucólisis , Fosfofructoquinasa-1/metabolismo , Sinaptosomas/metabolismo , Adenosina Monofosfato/metabolismo , Amoníaco/metabolismo , Animales , Células Cultivadas , Metabolismo Energético , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Iones , Concentración Osmolar , Fósforo/metabolismo , Ratas , Rotenona/farmacologíaRESUMEN
Changes in cellular energetic and metabolic parameters were analyzed at several time points during apoptosis of differentiated PC12 cells following removal of nerve growth factor (NGF). As approximately 60% of the population died during the period of study (24 h), most of the measured metabolic indicators declined over time. However, this decline paralleled the overall decrease in cellular viability, suggesting that, in individual cells, a compromised metabolic state occurred suddenly and very late in the death process. For example, when expressed as a function of viable cells, protein and RNA synthesis did not decrease until 24 h. Glucose utilization in live cells was never significantly reduced relative to control levels; lactate production decreased slightly within 4-8 h after NGF removal, but eventually rebounded to 122% of control levels by 24 h. ATP levels dropped 27% in an early predeath period, but then returned to near control levels (on a per-live-cell basis) once the population actively began to die. The ATP/ADP ratio remained at least 84% of control throughout. UTP/UDP and GTP/GDP ratios did not change significantly at any time point.
Asunto(s)
Apoptosis , Metabolismo Energético , Neuronas/fisiología , Animales , Supervivencia Celular , Células Clonales , Lactatos/biosíntesis , Ácido Láctico , Factores de Crecimiento Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/metabolismo , Nucleótidos/metabolismo , Células PC12 , ARN/metabolismo , RatasRESUMEN
The interaction of spermine, spermidine and putrescine with the Ca(2+)-binding protein, parvalbumin, was studied at pH 6 and 7, with the help of the intrinsic fluorescence properties of tryptophan and circular dichroic spectroscopy of the protein in the ultraviolet region. Polyamines bind to parvalbumin that is either Ca(2+)-free or partially saturated with Ca2+, as indicated by a change in the emission maximum and intensity of tryptophan fluorescence. The binding affinities for the interactions are about 4 mM, 8 mM and > 20 mM for spermine, spermidine and putrescine, respectively. No alterations in fluorescence properties were detected when the polyamines were added to fully Ca(2+)-bound parvalbumin. An increase in the ellipticity of the circular dichroic spectrum in the region where tryptophan absorbs was observed when polyamines were added to Ca(2+)-free parvalbumin. This finding indicates that polyamine binding affects the segment of the protein where tryptophan is located. Based on these results it is postulated that polyamines bind to the Ca(2+)-free or partially saturated parvalbumin and stabilize its structure.