RESUMEN
This work consists of an experimental determination of k0 and Q0 for 121Sb, 123Sb and 130Ba targets. Covariance analysis has been introduced to supply not only the overall uncertainties in these parameters but also their correlations. The irradiations were performed near the core of the IEA-R1 4.5 MW swimming-pool nuclear research reactor of the Nuclear and Energy Research Institute (IPEN-CNEN/SP), in São Paulo, Brazil. The epithermal neutron flux shape parameter, alpha, at the irradiation position is very close to zero, which favors to obtain Q0 values more accurately. Two irradiations were carried out in sequence, using two sets of samples: the first with bare samples and the second inside a Cd cover. The activity measurements were carried out in a previously calibrated HPGe gamma-ray spectrometer. The measurements were corrected for: saturation, decay time, cascade summing, geometry, self-attenuation, measuring time and mass. Standard sources of 152Eu, 133Ba, 60Co and 137Cs traceable to a 4πß-γ primary system were used to obtain the HPGe gamma-ray peak efficiency as a function of the energy. The experimental efficiency curve was performed by a fourth-degree polynomial fit, in the energy range of the standard sources, 121-1408 keV, it contains all correlations between points. For energies above 1408 keV, the efficiencies were obtained by the Monte Carlo Method. The covariance matrix methodology was applied to all uncertainties involved. The final values for k0 and Q0 were compared with the literature.
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Método de Montecarlo , BrasilRESUMEN
The energy cost of information processing is thought to be chiefly neuronal, with a minor fraction attributed to glial cells. However, there is compelling evidence that astrocytes capture synaptic K+ using their Na+/K+ ATPase, and not solely through Kir4.1 channels as was once thought. When this active buffering is taken into account, the cost of astrocytes rises by >200%. Gram-per-gram, astrocytes turn out to be as expensive as neurons. This conclusion is supported by 3D reconstruction of the neuropil showing similar mitochondrial densities in neurons and astrocytes, by cell-specific transcriptomics and proteomics, and by the rates of the tricarboxylic acid cycle. Possible consequences for reactive astrogliosis and brain disease are discussed.
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Astrocitos , Encefalopatías , Astrocitos/metabolismo , Humanos , Neuroglía , Neuronas/metabolismo , Sodio/metabolismoRESUMEN
In the present work, the determinations of k0 and Q0 for 74Se, 113In, 186W and 191Ir targets were performed. The irradiations were conducted near the core of the IEA-R1 4.5â¯MW swimming-pool nuclear research reactor of the Instituto de Pesquisas Energéticas e Nucleares (IPEN-CNEN/SP - Nuclear and Energy Research Institute), in São Paulo, Brazil. The irradiation position was chosen where the neutron spectrum shape parameter α is very close to zero. For this reason, the correction to be applied for the determination of Q0 is very close to one, thus improving the accuracy of the results. For each experiment, two irradiations were carried out in sequence: the first one with bare samples and the second with a cadmium cover around the samples. All partial uncertainties were considered, applying the covariance matrix methodology. The final results were compared with the literature.
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The key protein in the canonical Wnt pathway is ß-catenin, which is phosphorylated both in absence and presence of Wnt signals by different kinases. Upon activation in the cytoplasm, ß-catenin can enter into the nucleus to transactivate target gene expression, many of which are cancer-related genes. The mechanism governing ß-catenin's nucleocytoplasmic transport has been recently unvealed, although phosphorylation at its C-terminal end and its functional consequences are not completely understood. Serine 646 of ß-catenin is a putative CK2 phosphorylation site and lies in a region which has been proposed to be important for its nucleocytoplasmic transport and transactivation activity. This residue was mutated to aspartic acid mimicking CK2-phosphorylation and its effects on ß-catenin activity as well as localization were explored. ß-Catenin S6464D did not show significant differences in both transcriptional activity and nuclear localization compared to the wild-type form, but displayed a characteristic granular nuclear pattern. Three-dimensional models of nuclei were constructed which showed differences in number and volume of granules, being those from ß-catenin S646D more and smaller than the wild-type form. FRAP microscopy was used to compare nuclear export of both proteins which showed a slightly higher but not significant retention of ß-catenin S646D. Altogether, these results show that C-terminal phosphorylation of ß-catenin seems to be related with its nucleocytoplasmic transport but not transactivation activity.
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Transporte Activo de Núcleo Celular , Activación Transcripcional , beta Catenina/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Fosforilación , Homología de Secuencia de Aminoácido , beta Catenina/químicaRESUMEN
Brain tissue is highly dynamic in terms of electrical activity and energy demand. Relevant energy metabolites have turnover times ranging from milliseconds to seconds and are rapidly exchanged between cells and within cells. Until recently these fast metabolic events were inaccessible, because standard isotopic techniques require use of populations of cells and/or involve integration times of tens of minutes. Thanks to fluorescent probes and recently available genetically-encoded optical nanosensors, this Technology Report shows how it is now possible to monitor the concentration of metabolites in real-time and in single cells. In combination with ad hoc inhibitor-stop protocols, these probes have revealed a key role for K(+) in the acute stimulation of astrocytic glycolysis by synaptic activity. They have also permitted detection of the Warburg effect in single cancer cells. Genetically-encoded nanosensors currently exist for glucose, lactate, NADH and ATP, and it is envisaged that other metabolite nanosensors will soon be available. These optical tools together with improved expression systems and in vivo imaging, herald an exciting era of single-cell metabolic analysis.
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Neuronal activity is accompanied by a rapid increase in interstitial lactate, which is hypothesized to serve as a fuel for neurons and a signal for local vasodilation. Using FRET microscopy, we report here that the rate of glycolysis in cultured mice astrocytes can be acutely modulated by physiological changes in extracellular lactate. Glycolytic inhibition by lactate was not accompanied by detectable variations in intracellular pH or intracellular ATP and was not dependent of mitochondrial function. Pyruvate was also inhibitory, suggesting that the effect of lactate is not mediated by the NADH/NAD(+) ratio. We propose that lactate serves as a fast negative feedback signal limiting its own production by astrocytes and therefore the amplitude of the lactate surge. The inhibition of glucose usage by lactate was much stronger in resting astrocytes than in K(+)-stimulated astrocytes, which suggests that lactate may also help diverting glucose from resting to active zones.
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Astrocitos/efectos de los fármacos , Retroalimentación Fisiológica/efectos de los fármacos , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Ácido Láctico/farmacología , Adenosina Trifosfato/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Astrocitos/citología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Células Cultivadas , Corteza Cerebral/citología , Citocalasina B/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Líquido Extracelular/efectos de los fármacos , Líquido Extracelular/metabolismo , Concentración de Iones de Hidrógeno , Ácido Yodoacético/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Potasio/farmacología , Ionóforos de Protónes/farmacología , Rotenona/farmacologíaRESUMEN
Knowing how different cell types handle glucose should help to decipher how energy supply is adjusted to energy demand in the brain. Previously, the uptake of glucose by cultured brain cells was studied in real-time using fluorescent tracers and confocal microscopy. Here, we have adapted this technique to acute slices prepared from the rat cerebellum by means of multiphoton microscopy. The transport of the fluorescent glucose analogs 2NBDG and 6NBDG was several-fold faster in the molecular layer of the cerebellar cortex than in Purkinje cell somata and granule cells. After washout of free tracer, it became apparent that most phosphorylated tracer was located in Bergmann glia, which was confirmed by counterstaining with the glial marker sulforhodamine 101. The effective recovery of fluorescence after photobleaching showed that 2NBDG-P can diffuse horizontally across the molecular layer, presumably through gap junctions between Bergmann glial cells. Our main conclusion is that in acute cerebellar slices, the glucose transport capacity and glycolytic rate of Bergmann glia are several-fold higher than those of Purkinje cells. Given that the cerebellum is largely fueled by glucose and Purkinje neurons are estimated to spend more energy than Bergmann glial cells, these results suggest substantial shuttling of an energy-rich metabolite like lactate between glial cells and neurons.
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Cerebelo/metabolismo , Glucosa/metabolismo , Neuroglía/metabolismo , Células de Purkinje/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Animales , Transporte Biológico , Desoxiglucosa/análogos & derivados , Desoxiglucosa/metabolismo , Fluorescencia , Recuperación de Fluorescencia tras Fotoblanqueo , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucosa/análogos & derivados , Proteínas de Transporte de Glutamato en la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Proteínas Fluorescentes Verdes/genética , Técnicas In Vitro , Ratones , Ratones Transgénicos , Microscopía de Fluorescencia por Excitación Multifotónica , Ratas , Rodaminas , Factores de TiempoRESUMEN
Several diagnostic and therapeutic methods are based on the optical properties of lasers. In therapeutic applications, laser light is absorbed in a specific manner, whereas light is scattered, reflected, or transmitted from different structures. Improvements in laser technology allow new procedures and broaden the scope of applications for both diagnosis and therapy. The focus of laser application in Oral Medicine diagnosis should be early detection of oral squamous cell carcinoma. Novel modalities for the detection of oral malignancy are urgently needed, while others must be continuously improved. Optical coherence tomography and laser-induced fluorescence spectroscopy are currently being studied. In addition to diagnosis of non-malignant lesions, laser therapy has been used based upon the biological reactions and molecular wound healing mechanisms as an alternative for the treatment of a variety of oral soft tissue lesions. The aim of the present article is to review current knowledge and future perspectives of lasers in Oral Medicine.
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Terapia por Láser , Rayos Láser , Enfermedades de la Boca/diagnóstico , Enfermedades de la Boca/terapia , Predicción , HumanosRESUMEN
The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 microg protein, gels were incubated with 50, 100, or 250 microg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09%). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50% after incubation with 50 microg/mL and was completely abolished at 100 and 250 microg/mL of the extract. After incubation with 50 microg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50%. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 microg/mL of the extract. For MMP-2 the incubation with 100 or 250 microg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.
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Quemaduras Químicas/enzimología , Lesiones de la Cornea , Inhibidores Enzimáticos/farmacología , Quemaduras Oculares/inducido químicamente , Inhibidores de la Metaloproteinasa de la Matriz , Piperaceae/química , Animales , Córnea/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/aislamiento & purificación , Quemaduras Oculares/enzimología , Metaloproteinasas de la Matriz/metabolismo , Fitoterapia , Extractos Vegetales/farmacología , ConejosRESUMEN
The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 æg protein, gels were incubated with 50, 100, or 250 µg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09 percent). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50 percent after incubation with 50 µg/mL and was completely abolished at 100 and 250 µg/mL of the extract. After incubation with 50 µg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50 percent. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 µg/mL of the extract. For MMP-2 the incubation with 100 or 250 µg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.
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Animales , Conejos , Quemaduras Químicas/enzimología , Córnea/lesiones , Inhibidores Enzimáticos/farmacología , Quemaduras Oculares/inducido químicamente , Metaloproteinasas de la Matriz/antagonistas & inhibidores , Piperaceae/química , Córnea/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/aislamiento & purificación , Quemaduras Oculares/enzimología , Metaloproteinasas de la Matriz/metabolismo , Fitoterapia , Extractos Vegetales/farmacologíaRESUMEN
While glucose is constantly being "pulled" into the brain by hexokinase, its flux across the blood brain barrier (BBB) is allowed by facilitative carriers of the GLUT family. Starting from the microscopic properties of GLUT carriers, and within the constraints imposed by the available experimental data, chiefly NMR spectroscopy, we have generated a numerical model that reveals several hidden features of glucose transport and metabolism in the brain. The half-saturation constant of glucose uptake into the brain (K(t)) is close to 8 mM. GLUT carriers at the BBB are symmetric, show accelerated-exchange, and a K(m) of zero-trans flux (K(zt)) close to 5 mM, determining a ratio of 3.6 between maximum transport rate and net glucose flux (T(max)/CMR(glc)). In spite of the low transporter occupancy, the model shows that for a stimulated hexokinase to pull more glucose into the brain, the number or activity of GLUT carriers must also increase, particularly at the BBB. The endothelium is therefore predicted to be a key modulated element for the fast control of energy metabolism. In addition, the simulations help to explain why mild hypoglycemia may be asymptomatic and reveal that [glucose](brain) (as measured by NMR) should be much more sensitive than glucose flux (as measured by PET) as an indicator of GLUT1 deficiency. In summary, available data from various sources has been integrated in a predictive model based on the microscopic properties of GLUT carriers.
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Glucosa/metabolismo , Músculo Liso Vascular/metabolismo , Neuroglía/metabolismo , Animales , Transporte Biológico Activo/fisiología , Glucemia/metabolismo , Química Encefálica/fisiología , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Hipoglucemia/metabolismo , Modelos Neurológicos , Músculo Liso Vascular/citologíaRESUMEN
We set out to identify molecular mechanisms underlying the onset of necrotic Ca(2+) overload, triggered in two epithelial cell lines by oxidative stress or metabolic depletion. As reported earlier, the overload was inhibited by extracellular Ca(2+) chelation and the cation channel blocker gadolinium. However, the surface permeability to Ca(2+) was reduced by 60%, thus discarding a role for Ca(2+) channel/carrier activation. Instead, we registered a collapse of the plasma membrane Ca(2+) ATPase (PMCA). Remarkably, inhibition of the Na(+)/K(+) ATPase rescued the PMCA and reverted the Ca(2+) rise. Thermodynamic considerations suggest that the Ca(2+) overload develops when the Na(+)/K(+) ATPase, by virtue of the Na(+) overload, clamps the ATP phosphorylation potential below the minimum required by the PMCA. In addition to providing the mechanism for the onset of Ca(2+) overload, the crosstalk between cation pumps offers a novel explanation for the role of Na(+) in cell death.
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Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Sodio/metabolismo , Animales , ATPasas Transportadoras de Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Perros , Células HeLa , Humanos , Modelos Biológicos , Necrosis , Estrés Oxidativo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Oxidative stress releases intracellular calcium, which plays a pathogenic role in mammalian cell death. Here we report a search for the source of oxidative calcium in HeLa cells based on confocal epifluorescence microscopy. H(2)O(2) caused a rapid increase in cytosolic calcium, which was followed by mitochondrial Ca(2+) loading. Combined mitochondrial uncoupling with full depletion of thapsigargin-sensitive stores abrogated inositol 1,4,5-trisphosphate-mediated calcium release but failed to inhibit H(2)O(2)-induced calcium release, observation that was confirmed in MDCK cells. Prevention of peroxide-induced acidification with a pH clamp was also ineffective, discarding a role for endosomal/lysosomal Ca(2+)/H(+) exchange. Lysosomal integrity was not affected by H(2)O(2). Mature human erythrocytes also reacted to peroxide by releasing intracellular calcium, thus directly demonstrating the cytosolic source. Glutathione depletion markedly sensitized cells to H(2)O(2), an effect opposite to that achieved by DTT. Iron chelation was ineffective. In summary, our results show the existence of a previously unrecognized sulfhydryl-sensitive source of pathogenic calcium in the cytosol of mammalian cells.
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Calcio/metabolismo , Citosol/metabolismo , Ácido Egtácico/análogos & derivados , Células Epiteliales/metabolismo , Peróxido de Hidrógeno/farmacología , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular , Perros , Ácido Egtácico/farmacología , Retículo Endoplásmico/fisiología , Células Epiteliales/efectos de los fármacos , Espacio Extracelular/fisiología , Células HeLa , Humanos , Peróxido de Hidrógeno/antagonistas & inhibidores , Concentración de Iones de Hidrógeno , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Factores de TiempoRESUMEN
The molecular identity of K(+) channels involved in Ehrlich cell volume regulation is unknown. A background K(+) conductance is activated by cell swelling and is also modulated by extracellular pH. These characteristics are most similar to those of newly emerging TASK (TWIK-related acid-sensitive K(+) channels)-type of two pore-domain K(+) channels. mTASK-2, but not TASK-1 or -3, is present in Ehrlich cells and mouse kidney tissue from where the full coding sequences were obtained. Heterologous expression of mTASK-2 cDNA in HEK-293 cells generated K(+) currents in the absence intracellular Ca(2+). Exposure to hypotonicity enhanced mTASK-2 currents and osmotic cell shrinkage led to inhibition. This occurred without altering voltage dependence and with only slight decrease in pK(a) in hypotonicity but no change in hypertonicity. Replacement with other cations yields a permselectivity sequence for mTASK-2 of K(+) > Rb(+) Cs(+) > NH(4)(+) > Na(+) congruent with Li(+), similar to that for the native conductance (I(K, vol)). Clofilium, a quaternary ammonium blocker of I(K, vol), blocked the mTASK-2-mediated K(+) current with an IC(50) of 25 microm. The presence of mTASK-2 in Ehrlich cells, its functional similarities with I(K, vol), and its modulation by changes in cell volume suggest that this two-pore domain K(+) channel participates in the regulatory volume decrease phenomenon.
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Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio/química , Canales de Potasio/metabolismo , Animales , Northern Blotting , Carcinoma de Ehrlich , Cationes , Línea Celular , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Electrofisiología , Humanos , Concentración de Iones de Hidrógeno , Immunoblotting , Riñón/metabolismo , Ratones , Reacción en Cadena de la Polimerasa , Potasio/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Compuestos de Amonio Cuaternario/farmacología , ARN/metabolismo , ARN Mensajero/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
The effect of osmotic stress on sugar transport was investigated in Clone 9 epithelial cells, which express the glucose uniporter GLUT1, and in 3T3-L1 adipocytes, which express both GLUT1 and GLUT4. An acute hyperosmotic shock increased the uptake of sugars in both cell types. In Clone 9 cells, this was followed by a regulatory volume increase (RVI) response. Stimulation of transport was rapid and reversible, with half-lives (t 1/2) for stimulation of 2-deoxy-D-glucose uptake of 5.6 +/- 0.9 (n=6) and 22.7 +/- 1.5 (n=4) min for Clone 9 cells and adipocytes respectively. The effect was dose dependent, reaching a maximum at 1.1 osM of 2.9 +/- 0.1-fold (n=3) for Clone 9 cells and 8.2 +/- 0.8-fold (n=3) for adipocytes. In the latter, this stimulation correlated with translocation of the glucose transporter isoform GLUT4 to the cell surface and was not significantly different from that elicited by 160 nM insulin (7.6 +/- 1.2-fold, n=3). The effect of osmotic shock was not, however, influenced by inhibitors of either phosphoinositide 3-kinase (PI 3-kinase) (wortmannin, 250 nM) or of p38 mitogen-activated protein kinase (p38 MAP kinase) (SB203580, 20 microM), which reportedly prevent GLUT4 translocation and/or activation by insulin respectively. These inhibitors also had no effect on the stimulation of transport by osmotic shock in Clone 9 cells. However, in contrast to adipocytes, the effect of osmotic shock on glucose transport in Clone 9 cells reflected primarily a change in the intrinsic activity of cell surface transporters and there was only a minor change in their subcellular distribution as assessed by cell immunostaining or no change as assessed by surface biotinylation. These results indicate that the response of cells to osmotic shock can involve changes both in transporter activity and location. The signal transduction pathways involved include neither PI 3-kinase nor the classical, osmotically-activated component, p38 MAP kinase.
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Adipocitos/metabolismo , Células Epiteliales/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Presión Osmótica , Células 3T3 , Adipocitos/citología , Animales , Antimetabolitos/farmacocinética , Transporte Biológico/fisiología , Membrana Celular/metabolismo , Desoxiglucosa/farmacocinética , Células Epiteliales/citología , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Mamíferos , Ratones , Ratas , Solución Salina Hipertónica/farmacocinética , Sorbitol/farmacocinética , Sacarosa/farmacocinéticaRESUMEN
Whether a lethally injured mammalian cell undergoes necrosis or apoptosis may be determined by the early activation of specific ion channels at the cell surface. Apoptosis requires K+ and Cl- efflux, which leads to cell shrinking, an active phenomenon termed apoptotic volume decrease (AVD). In contrast, necrosis has been shown to require Na+ influx through membrane carriers and more recently through stress-activated non-selective cation channels (NSCCs). These ubiquitous channels are kept dormant in viable cells but become activated upon exposure to free-radicals. The ensuing Na+ influx leads to cell swelling, an active response that may be termed necrotic volume increase (NVI). This review focuses on how AVD and NVI become conflicting forces at the beginning of cell injury, on the events that determine irreversibility and in particular, on the ion fluxes that decide whether a cell is to die by necrosis or by apoptosis.
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Apoptosis , Animales , Canales de Cloruro/metabolismo , Transporte Iónico , Necrosis , Canales de Potasio/metabolismoRESUMEN
Necrosis, as opposed to apoptosis, is recognized as a nonspecific cell death that induces tissue inflammation and is preceded by cell edema. In non-neuronal cells, the latter has been explained by defective outward pumping of Na(+) caused by metabolic depletion or by increased Na(+) influx via membrane transporters. Here we describe a novel mechanism of swelling and necrosis; namely the influx of Na(+) through oxidative stress-activated nonselective cation channels. Exposure of liver epithelial Clone 9 cells to the free-radical donors calphostin C or menadione induced the rapid activation of an approximately 16-pS nonselective cation channel (NSCC). Blockage of this conductance with flufenamic acid protected the cells against swelling, calcium overload, and necrosis. Protection was also achieved by Gd(3+), an inhibitor of stretch-activated cation channels, or by isosmotic replacement of extracellular Na(+) with N-methyl-D-glucamine. It is proposed that NSCCs, which are ubiquitous although largely inactive in healthy cells, become activated under severe oxidative stress. The ensuing influx of Na(+) initiates a positive feedback of metabolic and electrolytic disturbances leading cells to their necrotic demise.