RESUMEN

We studied the effects of lidocaine and tetrodotoxin (TTX) on hypoxic changes in CA1 pyramidal neurons to examine the ionic basis of neuronal damage. Lidocaine (10 and 100 microM) and TTX (6 and 63 nM) delayed and attenuated the hypoxic depolarization and improved recovery of the resting and action potentials after 10 min of hypoxia. Lidocaine (10 and 100 microM) and TTX (63 nM) reduced the number of morphologically damaged CA1 cells and improved protein synthesis measured after 10 min hypoxia. Lidocaine (10 microM) attenuated the increase in intracellular sodium (181 vs. 218%) and the depolarization (-21 vs. -1 mV) during hypoxia but did not significantly attenuate the changes in ATP, potassium, or calcium measured at 10 min of hypoxia. Lidocaine (100 microM) attenuated the changes in membrane potential, sodium, potassium, ATP, and calcium during hypoxia. TTX (63 nM) attenuated the changes in membrane potential (-36 vs. -1 mV), sodium (179 vs. 226%), potassium (78 vs. 50%), and ATP (24 vs. 11%) but did not significantly attenuate the increase in calcium during hypoxia. These data indicate that the primary blockade of sodium channels can secondarily alter other cellular parameters. The hypoxic depolarization and the increase in intracellular sodium appear to be important triggers of hypoxic damage independent of their effect on cytosolic calcium; a treatment that selectively blocked sodium influx (lidocaine 10 microM) improved recovery. Our data indicate that selective blockade of sodium channels with a low concentration of lidocaine or TTX improves recovery after hypoxia by attenuating the rise in cellular sodium and the hypoxic depolarization. This blockade improves the resting and action potentials, histologic state, and protein synthesis of CA1 pyramidal neurons after 10 min of hypoxia to rat hippocampal slices. A higher concentration of lidocaine, which also improved ATP, potassium, and calcium concentrations during hypoxia was more potent. In conclusion, the depolarization and increased sodium concentration during hypoxia account for a portion of the neuronal damage after hypoxia independent of changes in calcium.

Asunto(s)

Hipocampo/metabolismo , Hipocampo/patología , Hipoxia/patología , Células Piramidales/metabolismo , Células Piramidales/patología , Bloqueadores de los Canales de Sodio , Adenosina Trifosfato/metabolismo , Anestésicos Locales/farmacología , Animales , Calcio/metabolismo , Citosol/metabolismo , Electrofisiología , Lidocaína/farmacología , Masculino , Potenciales de la Membrana/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Técnicas de Placa-Clamp , Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Sodio/metabolismo , Tetrodotoxina/farmacología

RESUMEN

Neuronal protein synthesis is inhibited in CA1 pyramidal neurons for many hours after ischemia, hypoxia or hypoglycemia. This inhibition precedes cell death, is a hallmark characteristic of necrotic damage and may play a key role in the death of vulnerable neurons after these insults. The sequence of events leading to this inhibition remains to be fully elucidated. The protein synthesis failure after 7.5 min anoxia/aglycemia in the rat hippocampal slice can be prevented by blocking N-methyl-D-aspartate receptors in a reduced calcium environment during the insult. In this study, we demonstrate that N-methyl-D-aspartate exposure directly causes a dose-dependent, receptor-mediated and prolonged protein synthesis inhibition in CA1 pyramidal neurons. The free radical scavenger Vitamin E significantly attenuates this damage due to low concentrations of N-methyl-D-aspartate (10 microM). Free radical generation by xanthine/xanthine oxidase (XOD) can directly damage protein synthesis in neurons of the slice. Vitamin E, ascorbic acid and N-acetylcysteine can each prevent the damage due to anoxia/aglycemia and to higher concentrations of N-methyl-D-aspartate (50 microM), provided calcium levels are reduced concomitantly. These findings indicate that both free radicals and calcium play a role in the sequence of events leading to protein synthesis failure after energetic stress like anoxia/aglycemia. They further suggest that the mechanism by which N-methyl-D-aspartate receptor activation damages protein synthesis involves free radical generation.

Asunto(s)

Antagonistas de Aminoácidos Excitadores/farmacología , Hipoglucemia/fisiopatología , Hipoxia/fisiopatología , N-Metilaspartato/farmacología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/efectos de los fármacos , Animales , Maleato de Dizocilpina/farmacología , Depuradores de Radicales Libres/farmacología , Radicales Libres/efectos adversos , Radicales Libres/antagonistas & inhibidores , Radicales Libres/metabolismo , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-Dawley , Vitamina E/farmacología

RESUMEN

BACKGROUND AND PURPOSE: Thiopental has been shown to protect against cerebral ischemic damage; however, it has undesirable side effects. We have examined how thiopental alters histological, physiological, and biochemical changes during and after hypoxia. These experiments should enable the discovery of agents that share some of the beneficial effects of thiopental. METHODS: We made intracellular recordings and measured ATP, sodium, potassium, and calcium concentrations from CA1 pyramidal cells in rat hippocampal slices subjected to 10 minutes of hypoxia with and without 600 micromol/L thiopental. RESULTS: Thiopental delayed the time until complete depolarization (21+/-3 versus 11+/-2 minutes for treated versus untreated slices, respectively) and attenuated the level of depolarization at 10 minutes of hypoxia (-33+/-6 versus -12+/-5 mV). There was improved recovery of the resting potential after 10 minutes of hypoxia in slices treated with thiopental (89% versus 31% recovery). Thiopental attenuated the changes in sodium (140% versus 193% of prehypoxic concentration), potassium (62% versus 46%), and calcium (111% versus 197%) during 10 minutes of hypoxia. There was only a small effect on ATP (18% versus 8%). The percentage of cells showing clear histological damage was decreased by thiopental (45% versus 71%), and thiopental improved protein synthesis after hypoxia (75% versus 20%). CONCLUSIONS: Thiopental attenuates neuronal depolarization, an increase in cellular sodium and calcium concentrations, and a decrease in cellular potassium and ATP concentrations during hypoxia. These effects may explain the reduced histological, protein synthetic, and electrophysiological damage to CA1 pyramidal cells after hypoxia with thiopental.

Asunto(s)

Hipocampo/efectos de los fármacos , Hipnóticos y Sedantes/uso terapéutico , Hipoxia Encefálica/prevención & control , Fármacos Neuroprotectores/uso terapéutico , Células Piramidales/efectos de los fármacos , Tiopental/uso terapéutico , Potenciales de Acción/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Fenómenos Bioquímicos , Bioquímica , Isquemia Encefálica/prevención & control , Calcio/metabolismo , Electrofisiología , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/fisiopatología , Hipnóticos y Sedantes/administración & dosificación , Hipnóticos y Sedantes/efectos adversos , Hipoxia Encefálica/metabolismo , Hipoxia Encefálica/patología , Hipoxia Encefálica/fisiopatología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Neuronas/fisiología , Fármacos Neuroprotectores/administración & dosificación , Potasio/metabolismo , Biosíntesis de Proteínas , Proteínas/efectos de los fármacos , Células Piramidales/metabolismo , Células Piramidales/patología , Células Piramidales/fisiología , Ratas , Ratas Sprague-Dawley , Sodio/metabolismo , Tiopental/administración & dosificación , Tiopental/efectos adversos

RESUMEN

Protein synthesis is an extremely important cell function and there is now good evidence that changes in synthesis play important roles both in neuronal cell damage from ischemic insults and in neural plasticity though the mechanisms of these effects are not at all clear. The brain slice, and particularly the hippocampal slice, is an excellent preparation for studying these effects although, as with all studies on slices, caution must be exercised in that regulation in the slice may be different from regulation in vivo. Studies on neural tissue need to take into account the heterogeneity of neural tissue as well as the very different compartments within neurons. Autoradiography at both the light and electron microscope levels is a very powerful method for doing this. Successful autoradiography depends on many factors. These include correct choice of precursor amino acid, mechanisms for estimating changes in the specific activity of the precursor amino acid pool, and reliable methods for quantitation of the autoradiographs. At a more technical level these factors include attention to detail in processing tissue sections so as to avoid light contamination during exposure and developing and, also, appropriate choices of the various parameters such as exposure time and section thickness. The power of autoradiography is illustrated here by its ability to discern effects of ischemia and of plasticity-related neural input on distinct cell types and also in distinct compartments of neurons. Ischemia inhibits protein synthesis in principal neurons but activates synthesis in other cell types of the brain slice. Plasticity-related neural input immediately enhances protein synthesis in dendrites but does not affect cell bodies.

Asunto(s)

Hipocampo/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/metabolismo , Animales , Autorradiografía/métodos , Cicloheximida/farmacología , Disección , Cobayas , Hipocampo/citología , Hipocampo/ultraestructura , Técnicas In Vitro , Leucina/metabolismo , Microscopía Electrónica/métodos , Neuronas/citología , Neuronas/ultraestructura , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Tritio

RESUMEN

Tamoxifen, the major adjuvant drug treatment for estrogen-dependent breast cancer, has been shown previously to affect both estrogen-dependent and calcium/calmodulin-dependent pathways. In the current study, we developed an in vitro slice system to study the effects of tamoxifen on ATP levels in hypothalamic (HTH) and preoptic areas (POA) of the rat brain. Baseline data showed that, following a 2-h incubation, HTH and POA slices had comparable ATP levels to hippocampal slices, a system used extensively by researchers examining the metabolic responsiveness of the hippocampal region (HPC) of the brain. HTH-POA slice ATP levels remained steady for 2, 4 and 6 h, but fell to 11% of initial levels by 12 h. Neurons from HTH-POA slices incubated for 4 h appeared healthy and demonstrated robust protein synthesis as measured autoradiographically by incorporation of [3H]leucine. We explored the effects of tamoxifen (TAM), fluphenazine (FLU) and estradiol (E2) on ATP levels in HTH and POA slices. The effects of TAM were complex: a 4-h incubation with 10-6 M TAM led to decreased ATP levels in HTH (but not POA), and a 4-h incubation with 10-8 M led to increased ATP levels in POA (but not HTH); a 15-min exposure to 10-6 M TAM decreased ATP levels in POA (but not HTH) slices, while the exposure of slices to the lower concentration of TAM was without effect in either area. As with higher concentrations of TAM, 4-h incubation with 10-6 M FLU decreased ATP levels in HTH (but not POA), while incubation with E2 did not affect slice ATP levels. These data are consistent with the hypothesis that both TAM and FLU alter ATP levels in HTH slices via calmodulin- or calcium-mediated processes.

Asunto(s)

Adenosina Trifosfato/metabolismo , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Flufenazina/farmacología , Hipotálamo/metabolismo , Ovariectomía , Área Preóptica/metabolismo , Tamoxifeno/farmacología , Animales , Femenino , Hipotálamo/efectos de los fármacos , Técnicas In Vitro , Área Preóptica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Factores de Tiempo

RESUMEN

Extracellular pH modulates the function of the N-methyl-D-aspartate (NMDA) receptor, which may influence pathophysiological responses to glutamate. While damage due to oxygen and glucose deprivation or glutamate exposure is attenuated by acidification of the incubating medium of cultured neurons, neuron damage is enhanced in vivo following ischemia in hyperglycemic animals. A persistent inhibition of protein synthesis (to less than 5% of normoxic levels) is a reliable index of damage to neurons both in vivo and in the rat hippocampal slice. We explored the influence of extracellular pH and calcium manipulation on protein synthesis inhibition and energy failure due to anoxia/aglycemia or exposure to N-methyl-D-aspartate in the rat hippocampal slice. Moderate acidification of the medium during anoxia/aglycemia did not reduce the damage to protein synthesis in hippocampal neurons (9% of normoxic levels) and did not alter basal ATP levels or the rate of ATP depletion during anoxia/aglycemia. However, when calcium levels were lowered during the acidification and following the anoxia/aglycemia, protein synthesis was almost completely protected (84% of normoxic levels). Calcium reduction itself also attenuated the protein synthesis inhibition due to anoxia/aglycemia (to 55.6% of normoxic controls), but the protection was not as complete. In contrast, moderate acidification of the medium significantly reduced the damage to protein synthesis due to a brief exposure to NMDA (37% of control with NMDA, 78.9% of control with acidification during NMDA), even in the presence of extracellular calcium. Alkalinization of the medium exacerbated the protein synthesis inhibition following anoxia/aglycemia, and significantly reduced basal ATP levels (to 52% of normoxic control levels). Thus, pHo changes influence neuronal metabolism and response to anoxia/aglycemia. In addition, while acidification can reduce the excitotoxic damage caused by direct exposure to NMDA, it cannot reduce damage due to anoxia/aglycemia unless calcium is lowered concomitantly. Thus, both NMDA receptor activation and calcium are involved in the damage due to oxygen and glucose deprivation in the slice.

Asunto(s)

Calcio/metabolismo , Espacio Extracelular/metabolismo , Glucosa/deficiencia , Hipocampo/metabolismo , Hidrógeno/metabolismo , Hipoxia/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Acidosis/metabolismo , Ácidos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Masculino , N-Metilaspartato/farmacología , Ratas , Ratas Sprague-Dawley

RESUMEN

Anesthetics attenuate ischemic damage and so are often not used when preparing hippocampal slices for studies of ischemic or anoxic damage. In this study, we tested whether halothane, ether or isoflurane, when used briefly during slice preparation, have persistent effects on slice ATP metabolism, protein synthesis or morphology. We also tested the effects of anoxia with and without glucose on these slices. Five minutes of anoxia without glucose (anoxia-aglycemia) caused a dramatic depletion of ATP to less than 22% of control levels, a persistent inhibition of neuronal protein synthesis to less than 10% of control rates and severe morphological damage. Slices prepared using volatile anesthetics showed the same degree of damage due to anoxia-aglycemia, when compared with untreated tissue. In contrast, 5 min anoxia caused a 40% decrease in ATP levels in untreated tissue, but did not damage protein synthesis or morphology. While isoflurane-treated tissue responded identically to anoxia as untreated tissue, both halothane and ether prevented the anoxic ATP fall. These findings suggest that while halothane and ether may have persistent effects on slice responses to anoxia, isoflurane is a good candidate anesthetic for slice preparation procedures.

Asunto(s)

Anestésicos por Inhalación/farmacología , Metabolismo Energético/efectos de los fármacos , Glucosa/farmacología , Hipocampo/efectos de los fármacos , Hipoxia Encefálica/tratamiento farmacológico , Proteínas del Tejido Nervioso/biosíntesis , Análisis de Varianza , Animales , Éter/farmacología , Halotano/farmacología , Hipocampo/metabolismo , Hipoxia Encefálica/metabolismo , Técnicas In Vitro , Isoflurano/farmacología , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

Following 5 min in vitro ischemia, total protein synthesis is dramatically and persistently inhibited in neurons in the rat hippocampal slice. This model system was used to explore the responses of individual proteins to this irreversible insult. In vitro ischemia inhibited new protein synthesis of most proteins analyzed; however, the synthesis of a 68/70 kDa protein was substantially stimulated for the first hour after ischemia. By 3 hr postischemia, its synthesis rates were depressed to 60% of control rates. Although the total amounts of most proteins were not significantly depleted for the first few hours after ail ischemic episode, there were several notable exceptions. The levels of HSC73, a constitutively expressed member of the 70 kDa stress protein family, were reduced after in vitro ischemia. In addition, MAP-2 (microtubule-associated protein-2) and alpha-tubulin were depleted in the early hours after the insult, with MAP-2 exhibiting a detectable depletion earlier than tubulin. In contrast, the levels and distribution of a 68 kDa neurofilament protein localized to CA3 pyramidal neurons in the slice, apparently distinct from the band whose new synthesis was stimulated, were not affected by the 5 min in vitro ischemia insult. Thus, the responses of individual proteins to ischemia varied considerably, These individual responses could play an important role in the damage mechanism that is initiated in response to in vitro ischemia.

Asunto(s)

Isquemia Encefálica/metabolismo , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Isquemia Encefálica/patología , Colorantes , Proteínas del Citoesqueleto/metabolismo , Densitometría , Proteínas de Choque Térmico/metabolismo , Hipocampo/patología , Técnicas In Vitro , Masculino , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Plata , Factores de Tiempo

RESUMEN

We studied Cl-/HCO3- exchange function in acutely dissociated single hippocampal neurons from adult and fetal day 18 rat using a fluorescent intracellular pH (pHi) indicator dye. The presence of Cl-/HCO3- exchange activity was assayed by observing the elevation in pHi upon acute reversal of the Cl- gradient. Resting intracellular pH in acutely dissociated neurons of both adult and fetal tissue was significantly higher than that of cultured fetal hippocampal neurons (day 10-12 in culture). Acute removal of extracellular Cl- caused a rapid and reversible increase in pHi by 0.25 pH units in adult neurons but had virtually no effect in similarly dissociated fetal neurons. Cl-/HCO3- exchange activity was also undetectable in fetal cultured hippocampal neurons. The mRNA for the anion exchanger AE3 is expressed abundantly in adult rodent neurons. AE3 is a potential candidate molecule for the observed Cl-/HCO3- exchange activity. In situ hybridization was used to monitor expression of the AE3 gene in these two age groups. We found that both adult and fetal neurons express AE3 mRNA. These results indicate that AE3 may not function as a Cl-/HCO3- exchanger in fetal neurons, in contrast to its possible role in the adult brain.

Asunto(s)

Envejecimiento/metabolismo , Bicarbonatos/metabolismo , Cloruros/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Equilibrio Ácido-Base/fisiología , Animales , Femenino , Colorantes Fluorescentes , Hipocampo/citología , Hipocampo/embriología , Concentración de Iones de Hidrógeno , Hibridación in Situ , Intercambio Iónico , Embarazo , Ratas , Ratas Sprague-Dawley , Espectrometría de Fluorescencia

RESUMEN

A recently-developed semiconductor-based instrument, the silicon microphysiometer, allows for realtime, sensitive quantification of cellular metabolism in small numbers of cultured cells with relative case. This is accomplished by detecting the extrusion into the extracellular space of acidic metabolic products of glycolysis, respiration, and ATP hydrolysis, including lactic acid, CO2, and protons. In the present report, we use microphysiometry to observe that glucocorticoids inhibit metabolic rate (as assessed indirectly by a change in the extracellular acidification rate) in fibroblasts (minimal effective dose of 1 nM of corticosterone), whereas 1 microM each estradiol, progesterone and testosterone failed to do so. We suggest that this inhibition of metabolism is secondary to the well-established inhibition of glucose transport and of protein synthesis in fibroblasts by glucocorticoids.

Asunto(s)

Ácidos/metabolismo , Endocrinología/instrumentación , Glucocorticoides/farmacología , Animales , Células Cultivadas , Espacio Extracelular/química , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glucosa/metabolismo , Glucólisis , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , L-Lactato Deshidrogenasa/metabolismo , Microcomputadores , Ratas , Esteroides/farmacología

RESUMEN

Stress accelerates the growth of certain types of tumors. Here we report a possible metabolic mechanism underlying this phenomenon. Some early features of transformation include increased number of glucose transporters and greatly enhanced rates of glucose uptake; this adaptation accommodates the vast energy demands needed for neoplastic growth. In contrast, glucocorticoids, a class of steroid hormones secreted during stress, inhibit glucose transport in various tissues; this is one route by which circulating glucose concentrations are raised during stress. We reasoned that should transformed cells become resistant to this inhibitory action of glucocorticoids, such cells would gain preferential access to these elevated concentrations of glucose. In agreement with this, we observed that Fujinami sarcoma virus-transformed fibroblasts became resistant to this glucocorticoid action both in vitro and in the rat. As a result, under conditions where glucocorticoids exerted catabolic effects upon nontransformed fibroblasts (inhibition of metabolism and ATP concentrations), the opposite occurred in the virally transformed cells. We observe that this glucocorticoid resistance upon transformation cannot be explained by depletion of glucocorticoid receptors; previous studies have suggested that transformation causes an alteration in trafficking of such receptors. Because of this resistance of transformed fibroblasts to the inhibitory effects of glucocorticoids upon glucose transport, glucose stores throughout the body are, in effect, preferentially shunted to such tumors during stress.

Asunto(s)

Adenosina Trifosfato/metabolismo , Transformación Celular Neoplásica , Desoxiglucosa/metabolismo , Sarcoma Experimental/patología , Estrés Fisiológico/fisiopatología , Adrenalectomía , Animales , Transporte Biológico , Radioisótopos de Carbono , Dexametasona/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Retroviridae/genética , Retroviridae/patogenicidad , Sarcoma Experimental/metabolismo , Sarcoma Experimental/microbiología

RESUMEN

Increasing evidence implicates glutamate receptor over-stimulation in the neurotoxicity associated with a host of metabolic insults, including seizures and hypoxia-ischemia. To begin to understand more completely the role of energy metabolism in the mechanism of neuron death following excitatory amino acid exposure, we investigated the effects of kainic acid exposure on metabolic rate in cultured hippocampal cells using a recently developed silicon microphysiometer. The device gives a continual real-time measure of metabolism in relatively small numbers of cells, as assessed by efflux of protons generated at least in part by ATP hydrolysis and lactic acid production. In the first half of this report, we characterize the feasibility of using this device for measuring cellular metabolism in hippocampal cultures. Metabolic rate in both astrocytes and neurons was readily detectable, with a high signal-to-noise ratio. The rate was proportional to the number of cells and was sensitive to metabolic enhancement or depression. We then utilized this device to study metabolic responses to the excitotoxin kainic acid. We observed a receptor-mediated, dose-dependent increase in metabolic rate upon stimulation by kainic acid, with an EC50 of approximately 100 microM. Exposure to toxic levels of kainic acid for 10 min produced an initial elevation (for 2 hr) in metabolic rate and then a gradual decline in metabolism over the next 8 hr that preceded a measurable loss of cell viability. This study further delineates a time window for the onset of kainic acid-induced damage. The results clearly show the feasibility of using silicon microphysiometry for assessing metabolism of brain cultures and for exploring the relationship between metabolism and synaptic activation.

Asunto(s)

Hipocampo/metabolismo , Neurobiología/métodos , Neurotoxinas/farmacología , Animales , Células Cultivadas , Cianuros/farmacología , Relación Dosis-Respuesta a Droga , Glucosa/farmacología , Hipocampo/citología , Ácido Kaínico/farmacología , Neurobiología/instrumentación , Oxidación-Reducción/efectos de los fármacos , Silicio

RESUMEN

Regulation of intracellular pH (pHi) in single cultured rat hippocampal neurons was investigated using the fluorescent pHi indicator dye bis-carboxyethylcarboxyfluorescein. Resting pHi was dependent on the presence of bicarbonate and external Na+ but was not altered significantly by removal of Cl- or treatment with the anion exchange inhibitor diisothiocyanatostilbene-2,2'-disulfonate. Recovery of pHi from acute acid loading was due, in large part, to a pharmacologically distinct variant of the Na+/H+ antiporter. In nominally HCO3(-)-free solutions, this recovery exhibited a saturable dose dependence on extracellular Na+ (Km = 23-26 mM) or Li+. The antiporter was activated by decreasing pHi and was unaffected by collapse of the membrane potential with valinomycin. Like the Na+/H+ antiporter described in other cell systems, the hippocampal activity was inhibited by harmaline, but in sharp contrast, neither amiloride nor its more potent 5-amino-substituted analogues were able to prevent the recovery from an acid load. These data indicate that Na(+)-dependent mechanisms dominate pHi regulation in hippocampal neurons and suggest a role for a novel variant of the Na+/H+ antiporter.

Asunto(s)

Amilorida/farmacología , Proteínas Portadoras/fisiología , Hipocampo/química , Neuronas/química , Animales , Bicarbonatos , Proteínas Portadoras/efectos de los fármacos , Células Cultivadas , Harmalina/farmacología , Hipocampo/citología , Concentración de Iones de Hidrógeno , Ouabaína/farmacología , Ratas , Intercambiadores de Sodio-Hidrógeno

RESUMEN

The rat hippocampal slice was developed as a model for investigating the effects of ischemia on protein synthesis in different cell types, as synthesis is an early functional indicator of cell damage. Five min of in vitro ischemia inhibited protein synthesis in CA1 pyramidal and subicular neurons 3 h later, despite recovery of the energy charge. Morphology of these neurons was also affected. In contrast, glia and capillary endothelial cells showed increased synthesis at this time point, and no apparent structural changes. Exposure of slices to buffer lacking calcium and containing the non-competitive NMDA receptor blocker ketamine, during the 5 min ischemia, prevented both the inhibition of protein synthesis and the morphologic changes in the neurons. However, if buffer only lacked calcium, or only contained ketamine, both forms of ischemic damage occurred. Thus, the neuronal protein synthesis inhibition and the impaired morphology appear to be mediated by either extracellular calcium or NMDA receptor activation. In contrast to the neurons, the ischemia-induced stimulation of protein synthesis in glia and capillary endothelial cells was not affected by the above treatments, indicating that neither NMDA receptor activation nor extracellular calcium is necessary for this effect.

Asunto(s)

Calcio/fisiología , Hipocampo/irrigación sanguínea , Isquemia/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Neurotransmisores/fisiología , Animales , Hipocampo/metabolismo , Hipocampo/fisiopatología , Técnicas In Vitro , Masculino , Ratas , Ratas Endogámicas , Receptores de N-Metil-D-Aspartato