RESUMEN
Dorsal root ganglion neurons express functional AMPA and kainate receptors near their central terminals. Activation of these receptors causes a decrease in glutamate release during action potential evoked synaptic transmission. Due to differences in kinetic properties and expression patterns of these two families of glutamate receptors in subpopulations of sensory neurons, AMPA and kainate receptors are expected to function differently. We used embryonic dorsal root ganglion (DRG) neurons maintained in culture to compare functional properties of kainate and AMPA receptors. Most DRG neurons in culture expressed kainate receptors and about half also expressed AMPA receptors. Most AMPA and kainate receptor-expressing DRG neurons were sensitive to capsaicin, suggesting involvement of these glutamate receptors in nociception. When activated by kainate, AMPA receptors were capable of driving a sustained train of action potentials while kainate receptors tended to activate action potential firing more transiently. Glutamate elicited more action potentials and a larger steady-state depolarization in neurons expressing both AMPA and kainate receptors than in neurons expressing only kainate receptors. Adding to their more potent activation properties, AMPA receptors recovered from desensitization much more quickly than kainate receptors. Activation of presynaptic receptors by low concentrations of kainate, but not ATPA, caused a tetrodotoxin-sensitive increase in the frequency of spontaneous EPSCs recorded in dorsal horn neurons. By recording synaptic pairs of DRG and dorsal horn neurons, we found that activation of presynaptic kainate and AMPA receptors decreased evoked glutamate release from terminals of DRG neurons in culture. Our data suggest that the endogenous ligand, glutamate, will cause a different physiological impact when activating these two types of non-NMDA glutamate receptors at central or peripheral nerve endings of sensory neurons.
Asunto(s)
Neuronas Aferentes/fisiología , Receptores AMPA/metabolismo , Receptores de Ácido Kaínico/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Capsaicina/farmacología , Células Cultivadas , Embrión de Mamíferos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiología , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Neuronas Aferentes/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Receptores AMPA/efectos de los fármacos , Receptores de Ácido Kaínico/efectos de los fármacosRESUMEN
The protective effects of various divalent cations against the irreversible damage of myocardium, a phenomenon termed "the Ca2+-paradox", were examined in the isolated perfused pigeon heart. All cations examined were added at a concentration of 200 micromol l(-1) in the "calcium-free" medium. In hearts perfused with low calcium, upon normal calcium repletion, the maximal recovery of the contractile tension (in the 2nd minute) was approximately 115% and the recovery obtained at the end of reperfusion was 81.5% (compared to the equilibration period value). From the other divalent cations examined, the presence of cobalt, nickel, manganese or barium during calcium depletion powerfully protected the pigeon heart. Upon calcium repletion, the maximal recovery of contractile tension was approximately 60%, 76.5%, 100% and 85%, the recovery estimated at the end of reperfusion was 40%, 12%, 70% and 53%, and the resting tension estimated at the end of reperfusion was 2.69+/-0.18 g, 6.40+/-0.50 g, 1.20+/-0.10 g and 1.90+/-0.10 g for cobalt, nickel, manganese and barium, respectively. On the contrary, strontium exerted no protective effects. The protective effects were also indicated by reduced total protein and lactate dehydrogenase activity release into the effluent perfusate and maintenance of electrical activity. The effectiveness of the added divalent cations (with the exception of strontium) showed a strong dependence upon their ionic radius. The most potent inhibitors of this phenomenon in the pigeon heart were the divalent cations having an ionic radius closer to the ionic radius of calcium. These results are discussed in terms of the possible mechanisms involved in the protective effects of these cations.
Asunto(s)
Calcio/metabolismo , Cationes Bivalentes/farmacología , Columbidae/fisiología , Corazón/efectos de los fármacos , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Animales , Cationes Bivalentes/química , Cationes Bivalentes/metabolismo , Columbidae/metabolismo , Electrocardiografía , Corazón/fisiología , L-Lactato Deshidrogenasa/metabolismo , Perfusión , Proteínas/metabolismoRESUMEN
Sensory neurons express purinergic P2X receptors on their central and peripheral terminals as well as their cell bodies. ATP activation of these receptors drives action potential firing and glutamate release with potentially important consequences for sensory function. Here we show ATP-gated currents activated in cultured embryonic dorsal root ganglion neurons have heterogeneity of time-courses comparable to those observed in different subpopulations of acutely dissociated adult dorsal root ganglion neurons. The distribution of time-courses across the population of cultured neurons is strongly influenced by culture conditions. Heterogeneity in ATP current kinetics occurs even though immunocytochemical staining reveals a relatively homogeneous and widespread expression of the P2X2 and P2X3 subunits. We show that the time-courses of ATP-gated currents recorded at the cell bodies are mirrored by the time-courses of transmitter release from the dorsal root ganglion nerve terminals, indicating similar P2X receptor properties on the soma and their associated terminals. Our results illustrate a functional heterogeneity of P2X receptor-mediated currents that is strongly influenced by external factors. This heterogeneity in current kinetics may have implications for neuronal function as it constrains the time-course of ATP-mediated modulation of neurotransmitter release at sensory nerve terminals.
Asunto(s)
Adenosina Trifosfato/farmacología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Animales , Células Cultivadas , Resistencia a Medicamentos , Conductividad Eléctrica , Embrión de Mamíferos , Ganglios Espinales/citología , Ácido Glutámico/metabolismo , Inmunohistoquímica , Cinética , Ratas , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X3 , Factores de TiempoRESUMEN
Cells from major types of gliomas, i.e. oligodendrogliomas and glioblastomas, are able to generate action potentials upon a current injection similar to neurons (Patt et al. (1996) Neuroscience, 71, 601-611; Labrakakis et al. (1997b) J. Neuropath. Exp. Neurol., 56, 243-254. Here, we report that activation of ionotropic glutamate receptors by the selective agonist, kainate, or by glutamate itself, depolarized the tumour cells in culture and living slices from tumour tissue, and can elicit volleys of action potentials, as recorded with the patch-clamp technique. Sixty-six percent of the glioblastoma cells, 44% of the astocytoma and 86% of the oligodendroglioma cells responded to glutamate and the specific agonist of AMPA/kainate receptors, kainate. The involvement of non-NMDA (N-methyl-D-aspartate) receptors is further supported by the observation that both kainate and glutamate currents were blocked by CNQX (6-cyano-7-nitroquinoxaline-2,3-dione). The receptor activation was accompanied by an increase in cytosolic Ca2+, as recorded with a fura-2 microfluorometric system. The Ca2+ elevation was mediated by the activation of Ca2+ channels due to membrane depolarization. The presence of voltage-gated Ca2+ channels was confirmed by patch-clamp experiments. Taken together, these findings imply that the electrophysiological properties of glioma cells are more reminiscent of those of neurons than of glial cells.
Asunto(s)
Glioma/metabolismo , Glioma/fisiopatología , Receptores de Glutamato/fisiología , Calcio/metabolismo , Canales de Calcio/metabolismo , Conductividad Eléctrica , Electrofisiología , Glioma/patología , Ácido Glutámico/farmacología , Humanos , Membranas Intracelulares/metabolismo , Activación del Canal Iónico/fisiología , Ácido Kaínico/farmacología , Concentración Osmolar , Potasio/fisiología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Glioma cells in acute slices and in primary culture, and glioma-derived human cell lines were screened for the presence of functional GABA(A) receptors. Currents were measured in whole-cell voltage clamp in response to gamma-aminobutyric acid (GABA). While cells from the most malignant glioma, the glioblastoma multiforme, did not respond to GABA, an inward current (under our experimental conditions with high Cl- concentration in the pipette) was induced in gliomas of lower grades, namely in 71% of oligodendroglioma cells and in 62% of the astrocytoma cells. Glioma cell lines did not express functional GABA(A) receptors, irrespective of the malignancy of the tumour they originate from. The currents elicited by application of GABA were due to activation of GABA(A) receptors; the specific agonist muscimol mimicked the response, the antagonists bicuculline and picrotoxin blocked the GABA-activated current and the benzodiazepine receptor agonist flunitrazepam augmented the GABA-induced current and the benzodiazepine inverse agonist DMCM decreased the GABA current. Cells were heterogeneous with respect to the direction of the current flow as tested in gramicidin perforated patches: in some cells GABA hyperpolarized the membrane, while in the majority it triggered a depolarization. Moreover, GABA triggered an increase in [Ca2+]i in the majority of the tumour cells due to the activation of Ca2+ channels. Our results suggest a link between the expression of GABA receptors and the growth of glioma cells as the disappearance of functional GABA(A) receptors parallels unlimited growth typical for malignant tumours and immortal cell lines.
Asunto(s)
Glioblastoma/química , Oligodendroglioma/química , Receptores de GABA-A/análisis , Receptores de GABA-A/fisiología , Antibacterianos , Calcio/farmacocinética , Bloqueadores de los Canales de Calcio/farmacología , Flunitrazepam/farmacología , Agonistas del GABA/farmacología , Moduladores del GABA/farmacología , Gramicidina , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Muscimol/farmacología , Técnicas de Placa-Clamp , Picrotoxina/farmacología , Potasio/farmacología , Células Tumorales Cultivadas/química , Verapamilo/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Oligodendrocyte precursor cells are purported to migrate over long distances into the various brain regions where they differentiate into oligodendrocytes and fulfill their appropriate tasks, i.e., myelination of axons. Here we characterize motile oligodendrocyte precursor cells in detail. Video-time lapse analysis was performed on isolated precursor cells in single cell cultures, in co-culture with cerebellar microexplants, and in living brain slices. Motility analysis of individual cells was combined with electrophysiological, immunological, and morphological characterizations. Translocation of the cell bodies was not continuous but occurred in waves. All motile cells exhibited a simple morphology and most, but not all, of them expressed the A2B5 epitope in vitro. Patch clamp analysis of the motile cells confirmed that they belong to the O-2A lineage. The percentage of motile cells, as well as their velocities, were enhanced on substrate-coated laminin in comparison to poly-L-lysine. Motility was not influenced by the presence of cerebellar microexplants. O-2A progenitor cells did not migrate strictly along neurite fascicles which were projected from the microexplants. Glial progenitor cells in situ also did not strictly migrate along the main direction of the axonal fibers of the corpus callosum but rather traversed the fibers with an overall direction toward the cortex. After Lucifer Yellow filling of the motile progenitor cells in situ, we could demonstrate that they were dye-coupled to yet unidentified cells of the corpus callosum.
Asunto(s)
Encéfalo/citología , Movimiento Celular/fisiología , Oligodendroglía/fisiología , Células Madre/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Cerebelo/citología , Electrofisiología , Inmunohistoquímica , Técnicas In Vitro , Laminina/farmacología , Potenciales de la Membrana , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
GABA(A) receptor activation in cerebellar granule cells induced a complex physiological response, namely the activation of a Cl- conductance in concert with a blockade of the resting K+ outward conductance (by 71% as compared to controls). Both responses were mediated by the activation of GABA(A) receptors, since they were both mimicked by the GABA(A) receptor agonist muscimol and antagonized by picrotoxin and bicuculline. A substantial decrease of the mean open time of single, outwardly rectifying K+ channels was triggered by GABA as revealed from cell-attached recordings; this finding implies that an intracellular pathway links GABA(A) receptors and K+ channels. Furthermore, this action of GABA is mediated through the cytoplasm, as experiments with the cell-attached patch-clamp technique show. GABA induced a prominent membrane depolarization ranging from 10 to 25 mV as revealed by current-clamp recordings of gramicidin (or nystatin) permeabilized patches, thus selecting conditions not to perturb the physiological Cl- gradient across the cell. Our findings imply that the GABA-activated Cl- current depolarized the membrane as described for immature neurons. The blockade of the resting K+ channel conductance acts in concert and both mechanisms lead to this substantial depolarizing event.
Asunto(s)
Bicuculina/farmacología , Cerebelo/fisiología , Canales de Cloruro/fisiología , Cloruros/metabolismo , Muscimol/farmacología , Neuronas/fisiología , Picrotoxina/farmacología , Bloqueadores de los Canales de Potasio , Receptores de GABA-A/fisiología , Ácido gamma-Aminobutírico/farmacología , Animales , Bario/farmacología , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Factores de TiempoRESUMEN
Cells from primary cultures of four glioblastomas (GB), three low-grade astrocytomas (A), and four low-grade oligodendrogliomas (O) were tested for the presence of neuroligand receptors linked to Ca2+ signalling by calcium imaging. Cells of days 3 to 21 in culture were incubated with 5 microM fluo-3-acetomethylester in a bath solution and stimulated with 0.1 mM ATP, 0.01 mM angiotensin II, bradykinin, histamine, norepinephrine, serotonin, and substance P for 15 s, with 0.01 mM glutamate and 50 mM K+ for 30 s. Changes in the Ca2+ concentration were measured with a confocal laser scanning microscope. In all glioma subtypes, the majority of cells showed Ca2+ responses after application of histamine (60% of cells tested in GB, 67% in A, 86% in O), bradykinin (66% in GB, 29% in A, 55% in O) and ATP (48% in GB, 70% in A, 47% in O). The other stimuli induced Ca2+ transients in a smaller proportion (between 33% and 2%) of the cells. Our study demonstrates that histamine, bradykinin and ATP are potent inducers of [Ca2+]i signals in gliomas.
Asunto(s)
Calcio/metabolismo , Neurotransmisores/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Adenosina Trifosfato/farmacología , Agonistas alfa-Adrenérgicos/farmacología , Angiotensina II/farmacología , Bradiquinina/farmacología , Colorantes Fluorescentes , Glioblastoma , Glucosa/farmacología , Histamina/farmacología , Humanos , Norepinefrina/farmacología , Oligodendroglioma , Potasio/farmacología , Serotonina/farmacología , Sustancia P/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Vasoconstrictores/farmacologíaRESUMEN
We studied the electrophysiological properties of cells from human glioblastomas obtained after surgery. The membrane currents were compared in cells of acute tissue slices and primary cultures using the whole cell mode of the patch-clamp technique. Very strikingly, in about a third of the tumor cells in situ and in vitro, depolarizing voltage steps elicited large, tetrodotoxin-sensitive inward currents with a threshold of about -30 mV, indicating the presence of voltage-gated sodium channels. In addition, three types of potassium currents, a delayed rectifying, an A-type, and an inward rectifying, were observed. Such a set of voltage-gated channels is characteristic for neurons. Indeed, in these glioblastoma cells, depolarizing current pulses in the current clamp mode were able to generate action potentials with properties similar to those observed in neurons. We interpret this finding as the ability of glioblastoma cells to acquire neuronlike properties but retain some glial features, since they still express markers typical for astrocytes and their precursors. The role of sodium channels in glioblastoma cells is unclear at this moment and needs further investigation. Our findings, however, imply that the tumor tissue can be intrinsically excitable and that neoplastic glial cells themselves may be an etiologic factor for epileptic seizures.
Asunto(s)
Potenciales de Acción/fisiología , Neoplasias Encefálicas/fisiopatología , Glioblastoma/fisiopatología , Oligodendroglioma/fisiopatología , Adolescente , Adulto , Anciano , Femenino , Humanos , Canales Iónicos/fisiología , Masculino , Persona de Mediana Edad , Células Tumorales CultivadasRESUMEN
One of the most common symptoms of patients with oligodendrogliomas is the high frequency of epileptic seizures. We thus studied the physiological properties of cells in six human oligodendrogliomas and two oligoastrocytomas obtained from surgical material. The majority of tumor cells in living brain slices can generate action potentials as recorded with the patch-clamp technique indicating that this tissue is dominated by electrically excitable cells. In cultures from the same material, the action potential generating cells prevail within the first days and are subsequently replaced by electrically inexcitable cells. From histopathological and immunohistochemical data, the histogenesis of human oligodendroglial tumor is still uncertain. Our physiological study has not settled the debate on the origin of these tumors but revealed important findings with regard to this question. Since action potential generating glial cells have not been described in situ so far their occurrence in oligodendroglial tumors implies that oligodendroglial tumor cells may belong to the neuronal cell lineage.
Asunto(s)
Neoplasias Encefálicas/patología , Neuronas/fisiología , Oligodendroglioma/patología , Adulto , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/ultraestructura , Electrofisiología , Femenino , Glioma/metabolismo , Glioma/patología , Glioma/ultraestructura , Humanos , Inmunohistoquímica , Masculino , Potenciales de la Membrana/fisiología , Persona de Mediana Edad , Neuronas/metabolismo , Neuronas/ultraestructura , Oligodendroglioma/metabolismo , Oligodendroglioma/ultraestructura , Técnicas de Placa-Clamp , Canales de Sodio/metabolismo , Células Tumorales CultivadasRESUMEN
Central neurocytoma is a rare brain tumor composed of small round synaptophysin-positive cells, suggesting a neuronal origin of these tumor cells. Glial properties are inferred, however, from the observation that the tumor cells exhibit a strong morphological similarity to oligodendroglioma cells and show an astrocytic differentiation in vitro. To test for neuronal or glial physiological properties, we studied cultured neurocytoma cells derived from a surgical specimen from a 44-year-old man, employing the patch-clamp technique. Early primary cultures were composed of morphologically unique bi- or multipolar cells which were positive for synaptophysin and negative for the astrocyte marker glial fibrillary acidic protein. In the majority of these cells, whole-cell membrane current recordings revealed physiological properties of neurons, i.e., a high density of Na+ currents, the capacity to generate action potentials, and the expression of inotropic neurotransmitter receptors. Metabotropic neurotransmitter receptors could be demonstrated by Ca2+ imaging techniques. The remaining bi- or multipolar cells and almost all cells in later culture stages and in vitro passage lacked these neuronal properties and showed physiological features characteristic of glial cells. We conclude that the major population of neurocytoma cells shows physiological properties of neurons and that with time in culture this population is replaced by electrically passive cells.