RESUMEN
Trigerminal ganglia of 4 adult albino mice of the NMRI outbred stock were examined by electron microscopy. In all animals, about 10% of the neurons contained intracisternal A particles. Isolated structures resembling intracisternal A particles could be detected in atleast 50% of the nerve cells and in a few Schwann cells. Budding at the cell surface and/or extracellular type-C particles were not observed. An intracerebrally transplanted mouse C1300 neuroblastoma was likewise studied. Most tumor cells exhibited large numbers of intracisternal A particles having the same ultrastructure as the particles in trigeminal neurons. In addition, budding and extracellular type-C particles were occasionally observed.
Asunto(s)
Cuerpos de Inclusión Viral/ultraestructura , Neuroblastoma/microbiología , Nervio Trigémino/ultraestructura , Animales , Ratones , Neoplasias Experimentales/microbiología , Virus Oncogénicos/ultraestructura , Retroviridae/ultraestructuraRESUMEN
Poly(A)+ RNA from C6-BU-1 rat glioma cells and rat astroglial cells induced isoleucine transport activity when injected into Xenopus laevis oocytes. The Na+-independent component of isoleucine transport was inhibited by leucine, phenylalanine and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) but neither by methylaminoisobutyric acid (MeAIB) nor lysine. A Km value of approx. 100 microM was determined for the Na+-independent transport of isoleucine. These data are in accordance with expression of a system L like transporter. By injection of size fractionated poly(A)+ RNA a length of approx. 1.9 kb was determined for the pertinent mRNA.
Asunto(s)
Encéfalo/metabolismo , Isoleucina/metabolismo , Oocitos/metabolismo , Sistemas de Transporte de Aminoácidos , Animales , Transporte Biológico , Proteínas Portadoras/metabolismo , Relación Dosis-Respuesta a Droga , Expresión Génica , Cinética , Microinyecciones , Poli A/genética , ARN Mensajero/aislamiento & purificación , ARN Mensajero/farmacología , Ratas , Ratas Wistar , Sodio/farmacología , Células Tumorales Cultivadas , Xenopus laevisRESUMEN
A variety of pathological conditions lead to swelling of astrocytes, which in turn stimulates ion release by activation of ion channels at the plasma membrane. In the present study, acridine orange and fluorescein isothiocyanate coupled to dextran (FITC-dextran) have been used to examine the effect of cell swelling on pH in acidic compartments of cultured astroglial cells. Both NH4Cl (2 mM) and chloroquine (10 microM), known to alkalinize acidic cellular compartments, led to the expected increase in acridine orange fluorescence intensity. Similar, albeit smaller, effects were elicited by a reduction of extracellular osmolarity (-80 mOsm) and treatment of the cells with glutamate (l mM), manoeuvres which enhanced cell volume. Determination of changes in the FITC-dextran fluorescence ratio (485/440 nm) allowed quantification of the pH changes in lysosomal compartments. Treatment with NH4Cl, reduced extracellular osmolarity and glutamate increased lysosomal pH by 0.65 +/- 0.07, 0.85 +/- 0.14 and 0.25 +/- 0.07, respectively. Measurement of cytosolic pH using 2',7',-bis-(2-carboxyethyl)-5- (and -6) carboxyfluorescein (BCECF) demonstrated a pronounced acidification following cell swelling, observed with both reduced extracellular osmolarity (by 0.23 +/- 0.05 pH units) and 1 mM glutamate (by 0.26 +/- 0.02 pH units). In conclusion, pH within lysosomes and possibly other acidic cellular compartments of astrocytes is increased by cell swelling, which may have important consequences for astrocyte function.
Asunto(s)
Astrocitos/metabolismo , Tamaño de la Célula , Líquido Intracelular/metabolismo , Naranja de Acridina/metabolismo , Cloruro de Amonio/farmacología , Animales , Cloroquina/farmacología , Dextranos/metabolismo , Espacio Extracelular/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Fluorometría , Ácido Glutámico/farmacología , Concentración de Iones de Hidrógeno , Soluciones Hipotónicas/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Concentración Osmolar , RatasRESUMEN
We set up recursion relations for calculating all even moments of the end-to-end distance of Porod-Kratky wormlike chains in D dimensions. From these moments we derive a simple analytic expression for the end-to-end distribution in three dimensions valid for all peristence lengths. It is in excellent agreement with Monte Carlo data for stiff chains and approaches the Gaussian random-walk distributions for low stiffness.
RESUMEN
The presence and the colocalization of the three intermediate filament proteins, glial fibrillary acidic protein (GFAP) and the marker of mesenchymal liver cells, vimentin, were studied by an immunofluorescence double-labeling technique in cultures of isolated rat perisinusoidal stellate cells (PSC) and hepatocytes, in cocultures of isolated PSC and hepatocytes as well as in cryostat sections of rat liver. GFAP and vimentin immunoreactivities were localized in cultured PSC which were identified by the presence of the cellular marker desmin, another intermediate filament protein, or the stellate morphology to be seen after staining for one of three intermediate filament proteins. Both GFAP and vimentin were strongly expressed in the perinuclear region and the cell processes of cultured PSC. Staining for GFAP highly coincided with that for vimentin or desmin in cultured PSC and with that for vimentin in the liver sections. Desmin-positive cells were always also GFAP-positive. However, of the GFAP-positive cells only an estimated 50% were found desmin-positive. The coexpression of desmin and GFAP in the same cells appear to be unique, since apparently it has not been previously reported for any other cell type. Almost all of the vimentin-positive cells in hepatocyte culture were also expressing GFAP. Since desmin was not found in all of the cultured cells with PSC morphology, GFAP is suggested as a more reliable marker for PSC than desmin.
Asunto(s)
Proteína Ácida Fibrilar de la Glía/análisis , Hígado/química , Hígado/citología , Animales , Anticuerpos/análisis , Biomarcadores/análisis , Células Cultivadas , Desmina/biosíntesis , Proteína Ácida Fibrilar de la Glía/inmunología , Proteínas de Filamentos Intermediarios/análisis , Microscopía Electrónica , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Vimentina/análisisRESUMEN
Transamination of branched-chain amino acids (BCAAs) catalyzed by the branched chain aminotransferase isoenzymes (BCATs) is believed to play an important role in nitrogen shuttling and excitatory neurotransmitter glutamate metabolism in brain. Recently, we have shown that the mitochondrial isoenzyme (BCATm) is the predominant form found in cultured astrocytes. In this study we used immunocytochemistry to examine the distribution of BCAT isoenzymes in cultured rat neurons and microglial cells. The cytoplasm of neurons displayed intense staining for the cytosolic isoenzyme (BCATc), whereas BCATm staining was not detectable in neurons. In contrast, microglial cells expressed BCATm in high concentration. BCATc appeared to be absent in this cell type. The second and committed step in the BCAA catabolic pathway is oxidative decarboxylation of the alpha-keto acid products of BCAT catalyzed by the branched-chain alpha-keto acid dehydrogenase (BCKD) enzyme complex. Because the presence of BCKD should provide an index of the ability of a cell to oxidize BCAA, we have also immunocytochemically localized BCKD in neuron and glial cell cultures from rat brain. Our results suggest ubiquitous expression of this BCKD enzyme complex in cultured brain cells. BCKD immunoreactivity was detected in neurons and in astroglial and microglial cells. Therefore, the expression of BCAT isoenzymes shows cell-specific localization, which is consistent with the operation of an intercellular nitrogen shuttle between neurons and astroglia. On the other hand, the ubiquitous expression of BCKD suggests that BCAA oxidation can probably take place in all types of brain cells and is most likely regulated by the activity state of BCKD rather than by its cell-specific localization.
Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Neuroglía/enzimología , Neuronas/enzimología , Transaminasas/metabolismo , Animales , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunohistoquímica , Isoenzimas/metabolismo , RatasRESUMEN
The first step in the catabolism of branched-chain amino acids (BCAA), reversible transamination, is catalyzed by one of the two isoforms of branched-chain amino acid aminotransferase (BCAT). The mitochondrial isoenzyme (BCATm) is widely distributed among tissues, whereas the cytosolic isoenzyme (BCATc) is restricted to only a few organs. Remarkably, BCATc is the prominent isoenzyme found in brain. The physiological significance of the subcellular compartmentation of BCAT is still not understood. To contribute to the elucidation of the cellular distribution of the two isoenzymes in brain, we used cultured rat glial cells in an immunocytochemical study to determine the pattern of BCAT isoenzyme expression by glial cells. Antiserum against BCATm generated a punctate staining pattern of astroglial cells, confirming the mitochondrial location of this isoenzyme. In contrast, the cytosol of galactocerebroside-expressing oligodendroglial cells and O2A progenitor cells displayed intense staining only for BCATc. In addition, subpopulations of astroglial cells exhibited BCATc immunoreactivity. The presence of BCATm in astrocytes is consistent with the known ability of these cells to oxidize BCAA. Furthermore, our results on BCATc provide support for the hypothesis that BCATs are also involved in nitrogen transfer from astrocytes to neurons.
Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Encéfalo/enzimología , Isoenzimas/metabolismo , Neuroglía/enzimología , Transaminasas/metabolismo , Animales , Western Blotting , Encéfalo/citología , Células Cultivadas , Galactosilceramidas/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Neuroglía/citología , Ratas , Ratas WistarAsunto(s)
Bucladesina/farmacología , Glioma/metabolismo , Células Híbridas/metabolismo , Tejido Nervioso/metabolismo , Neuroblastoma/metabolismo , Animales , Fusión Celular , Línea Celular , Corteza Cerebral/metabolismo , Corteza Cerebral/ultraestructura , Células Clonales , Citoplasma/ultraestructura , Transporte de Electrón , Células Híbridas/efectos de los fármacos , Ratones , Microscopía Electrónica , Tejido Nervioso/efectos de los fármacos , Tejido Nervioso/ultraestructura , RatasRESUMEN
Substance P stimulated the uptake of guanidinium in neuroblastoma X glioma hybrid cells and neuroblastoma cells but not in polyploid glioma cells. Guanidinium has previously been shown to pass the action potential Na+ channel in the two neuronal cell lines. Half-maximal stimulation was reached at 3 microM substance P and, with the hybrid cells, a saturation was seen above 10 microM. The analogue (D-Pro2,D-Trp7,9)-substance P, recently described as a substance P antagonist, caused a stimulation of guanidinium uptake comparable to that seen in the presence of substance P and did not inhibit the stimulation exerted by substance P. The pharmacological properties of the substance P-activated ion channel were investigated. Tubocurarine, phentolamine and propranolol blocked the substance P-stimulated guanidinium uptake with half-maximal inhibitory concentrations of 0.5, 5 and 50 microM. A similar characteristics has been found previously with the veratridine-activated Na+ channel in the cell lines investigated here. Peptides structurally related to substance P such as physalaemin and eledoisin, or others such as neurotensin, bradykinin, D-Ala2, Met5-enkephalinamide and ACTH(1-24) did not affect guanidinium uptake. In view of the high concentrations of substance P required for eliciting an effect in the cell lines, the involvement of specific receptors is questioned. A direct interaction of the peptide with the action potential Na+ channel is discussed.
Asunto(s)
Glioma/metabolismo , Neuroblastoma/metabolismo , Sustancia P/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Guanidina , Guanidinas/metabolismo , Células Híbridas/metabolismo , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Ratones , Ratas , Sodio/metabolismoRESUMEN
The effect of the nonapeptide bradykinin on the membrane potential of permanent cell lines from neural origin was studied. A hyperpolarizing response of 10-30 s duration was produced when bradykinin was iontophoretically applied onto polyploid rat glioma cells (clone C6-4-2). Starting from the resting membrane potential the peak value of the hyperpolarizing response was reached within 0.5-1.5 s. Then the potential returned more slowly to the original value. The hyperpolarization was associated with an approximately 50% decrease in membrane resistance. Neither Na+ nor Cl- seemed to be important for the hyperpolarizing response, since bradykinin elicited similar hyperpolarizations in cells exposed to media in which Na+ or Cl- were replaced by choline or isethionate, respectively. Ca2+ fluxes are unlikely to be involved, since the addition of D600 did not affect the hyperpolarizations induced by bradykinin. However, a 10-fold increase in the concentration of K+ in the medium reduced the amplitude of the hyperpolarization by 40 mV. Thus, the hyperpolarization induced by bradykinin is associated with decrease in membrane resistance which is likely to be caused by an increased K+-conductance. The glioma cells showed a desensitization upon repeated application of bradykinin. However, the sensitivity of the cells to bradykinin was restored after 3-8 min of incubation in the absence of bradykinin. Since an antagonist of bradykinin is not known, the specificity of the action of bradykinin is difficult to assess. Nevertheless, the hyperpolarizing response to bradykinin appears to be specific insofar as other peptides, i.e. lutoliberin, thyroliberin, neurotensin, substance P and apamin, exerted no effect on the membrane potential of the glioma cells. Bradykinin-elicited hyperpolarizations with characteristics similar to those described above could also be demonstrated in neuroblastoma X glioma hybrid cells, but not in multinucleated fibroblast cells.
Asunto(s)
Bradiquinina/farmacología , Glioma/fisiopatología , Células Híbridas/fisiología , Neuroblastoma/fisiopatología , Animales , Línea Celular , Células Híbridas/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Neoplasias Experimentales/fisiopatología , RatasRESUMEN
Both substance P and, to a lesser degree, serotonin activate cation permeability in neuroblastoma x glioma hybrid cells, as determined by measurement of [14C]guanidinium uptake. Serotonin potentiates the action of substance P by shifting the concentration-effect curve of substance P to the left. The EC50 value for the synergistic effect of serotonin was around 0.3 microM. Dopamine and noradrenaline displayed comparable activity, albeit only at 50 and 130 times higher concentrations, respectively. The order of potency of various substance P-analogues was not changed by serotonin, indicating that the specificity of the substance P site on the hybrid cells was not affected by serotonin. Various other neurotransmitters and peptides had no effect on the response of the hybrid cells to substance P. The serotonin receptor interacting with the substance P receptor may be classified as a 5-HT3-receptor since methysergide, cimetidine, and ketanserin were ineffective, but two inhibitors specific for 5-HT3-receptors, ICS 205-930 (3 alpha-tropanyl-1H-indole-3-carboxylic acid ester) and MDL 72222 (1 alpha H,3 alpha,5 alpha H-tropan-3-yl-3,5-dichlorobenzoate), blocked the effect of serotonin at nanomolar concentrations. However, the two serotonin antagonists might also be blocking the ion permeability, since at higher concentrations they fully inhibited the stimulation of guanidinium uptake by substance P or by substance P plus serotonin. The synergism between substance P and serotonin on the hybrid cells offers the opportunity to study the mechanism of interaction of neurotransmitter receptors on a permanent neuronal cell line.
Asunto(s)
Cationes/farmacocinética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Guanidinas/farmacocinética , Serotonina/farmacología , Sustancia P/farmacología , Células Tumorales Cultivadas/metabolismo , Catecolaminas/farmacología , Línea Celular , Interacciones Farmacológicas , Glioma , Guanidina , Indoles/farmacología , Neuroblastoma , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Tropanos/farmacología , Tropisetrón , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The co-localization of glutamine synthetase, glial fibrillary acidic protein, galactocerebroside and fibronectin was investigated by immunofluorescence double staining in primary cultures of dissociated brain cells from newborn mice. In cultures, grown in serum-free medium, containing dibutyryl-cAMP, glutamine synthetase was found in about half of the glial fibrillary acidic protein containing astroblasts. After addition of dexamethasone to the cultures, glutamine synthetase appeared also in another cell type, in which no glial fibrillary acidic protein, but fibronectin was detectable. This demonstrates that these cells, which are present in substantial amounts, are not of glial nature. In cultures treated with dibutyryl-cAMP no other cell type was found positive for fibronectin. For cultures grown in serum-containing medium lacking dibutyryl-cAMP, evidence was obtained that glutamine synthetase was induced by dexamethasone only in part of all cells staining for fibronectin. This suggests the presence of two different populations of fibronectin-positive cells. Oligodendrocytes, revealed by staining for galactocerebroside, never contained detectable amounts of glutamine synthetase, irrespective of the presence of dexamethasone. This also holds for cultures grown in serum-free medium containing dibutyryl-cAMP. However, under these conditions, oligodendroblasts are seen only very rarely. Our results demonstrate an unexpected heterogeneity in the cellular composition of primary cultures from newborn mouse brain.
Asunto(s)
Encéfalo/fisiología , Glutamato-Amoníaco Ligasa/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/enzimología , Células Cultivadas , Fibronectinas/metabolismo , Técnica del Anticuerpo Fluorescente , Galactosilceramidas/metabolismo , Proteína Ácida Fibrilar de la Glía , Histocitoquímica , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Ratones EndogámicosRESUMEN
The ability of astroglial cells to detoxify exogenously applied hydrogen peroxide (H2O2) was tested using astroglia-rich primary cultures derived from the brains of newborn rats. Incubation of astroglial cells with 100 microM H2O2 in the absence of glucose led to a 66% oxidation of the cellular glutathione within 30 s. Under these conditions, the cells were unable to re-establish the original high ratio of GSH/GSSG within 30 min of incubation. In contrast, if glucose was present the amount of GSSG produced on incubation with H2O2 was smaller (45% of total glutathione after 30 s) and the original ratio of GSH/GSSG was almost completely re-established within 10 min. If 100 microM H2O2 was applied, H2O2 disappeared from the incubation buffer with an apparent half-life of approximately 4 min. After 15 min of incubation, no H2O2 was detectable any more. The apparent half-life of H2O2 in the incubation buffer increased slightly but significantly with increasing concentration of H2O2 or when the cells were starved of glucose. A small reduction in the capacity of the cells to detoxify H2O2 was also observed after depletion of the glutathione content to 14% of control level by a 24 h pre-incubation of the cells in culture medium containing buthionine sulfoximine, an inhibitor of glutathione synthesis. Incubation of astroglial cells with mercaptosuccinate or 3-aminotriazole, inhibitors of glutathione peroxidase and catalase, respectively, only marginally reduced the rate of disappearance of H2O2 from the incubation buffer. In contrast, the rate of H2O2 clearance was strongly reduced in the presence of both inhibitors. These results demonstrate that glutathione peroxidase and catalase are involved in the detoxification of H2O2 by astroglial cells and that both enzymes are able to substitute for each other in the detoxification of H2O2.
Asunto(s)
Astrocitos/metabolismo , Catalasa/metabolismo , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Animales , Animales Recién Nacidos , Butionina Sulfoximina/farmacología , Catalasa/antagonistas & inhibidores , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Glucosa/farmacología , Glutatión/análogos & derivados , Glutatión/antagonistas & inhibidores , Glutatión/metabolismo , Disulfuro de Glutatión , Glutatión Peroxidasa/antagonistas & inhibidores , Semivida , Oxidación-Reducción , Ratas , Ratas WistarRESUMEN
Ion channels were studied in primary neuronal and in primary glial cultures from rat brain by measuring the uptake of guanidinium, an ion that can permeate the Na+ channel. Neuronal cells exhibit a veratridine-stimulated (EC50 30 microM) guanidinium uptake, which is blocked by tetrodotoxin (IC50 30nM). This demonstrates the presence of a voltage-dependent Na+ channel. In glial cells veratridine + scorpion toxin, but not veratridine or scorpion toxin alone can stimulate a tetrodotoxin-sensitive ion uptake, thus indicating a 'silent' Na+ channel in the glial cells. Phentolamine, propranolol and various local anesthetic drugs (e.g. tetracaine, dibucaine) blocked the two different kinds of Na+ channels in the two cell populations investigated.
Asunto(s)
Encéfalo/metabolismo , Canales Iónicos/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/metabolismo , Sodio/metabolismo , Anestésicos Locales/farmacología , Animales , Células Cultivadas , Ratas , Ratas Endogámicas , Tetrodotoxina/farmacología , Veratridina/farmacologíaRESUMEN
Primary cultures derived from the brains of newborn mice are quantitatively dominated by astroglial cells, but contain also oligodendroglial, phagocytic and ependymal cells. When confluent cultures are fed with glucose-free growth medium containing 25 mM sorbitol for 14 days, oligodendroglial, phagocytic and ependymal cells are eliminated from the culture, as judged by morphological and immunocytochemical criteria. The remaining cells stain positively for vimentin and glial fibrillary acidic protein and, therefore, can be considered as astroglial cells. Inoculation of freshly dissociated mouse brain cells in the absence of glucose in a sorbitol-containing medium is not possible; however, feeding of the cultures from day 2 on with sorbitol instead of glucose results in a pure astroglial culture at confluency. Therefore glucose-free growth medium supplemented with sorbitol can be considered a selective medium for astroglial cells in primary mouse glial cultures.
Asunto(s)
Astrocitos/citología , Encéfalo/citología , Glucosa/farmacología , Sorbitol/farmacología , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/análisis , Ratones , Ratones Endogámicos , Proteína Básica de Mielina/análisis , Vimentina/análisisRESUMEN
In order to contribute to the elucidation of the function of astrocyte glycogen in brain, studies on the fate of the glucosyl residues of glycogen were carried out on astroglia-rich primary cultures derived from the brains of newborn rats. On glucose deprivation astroglial cells rapidly deplete their glycogen. In contrast to the situation with hepatocytes, only lactate, but not glucose, is detectable in the medium surrounding the astroglial cells. Besides glucose, astroglial cultures can also use mannose as a substrate for the synthesis of glycogen and the generation of lactate. Although mannose-fed astroglial cells contain glucose-6-phosphate, they do not release a measurable amount of glucose into the culture medium. Instead of glucose the astroglial cells release high amounts of lactate into the culture medium. Gluconolactone or 2-deoxyglucose which prevent glycogen breakdown in astroglial cells after glucose deprivation, allow to discriminate between lactate generated from glycogen and lactate from other sources. The amount of lactate found in the medium in the absence of gluconolactone (or 2-deoxyglucose) exceeds the amount found in the presence of either compound by the lactate equivalents calculated to be contained in the cellular glycogen. In conclusion, glycogen in astrocytes can be considered as a store for lactate rather than for glucose.
Asunto(s)
Astrocitos/metabolismo , Glucógeno/metabolismo , Lactatos/metabolismo , Animales , Células Cultivadas , Glucosa/deficiencia , Hígado/citología , Hígado/metabolismoRESUMEN
The nonapeptide, bradykinin, elevated the level of cyclic GMP in two neural cell lines, neuroblastoma X glioma hybrid cells (clone 108CC15) and glioma cells (clone C6-4-2). In the hybrid cells the half-maximal stimulation occurred at 0.1 nM and the maximum was reached at 10 nM bradykinin. As soon as 30 s after the addition of bradykinin to the cultured cells, the intracellular concentration of cyclic GMP had increased maximally. The subsequent decline to the original level proceeded more slowly and lasted around 10 min. Hybrid cells incubated for 10 min in the presence of bradykinin and washed thereafter, did not respond at all to a subsequent 1 min challenge incubation with bradykinin. This nearly complete desensitization lasted for a period of 20 min. One hour after removal of bradykinin the original response to the peptide was restored. Modified and partial sequences of bradykinin were also investigated for their ability to induce the cyclic GMP response in the hybrid cells. Removal of amino acids from either terminus of bradykinin led to an almost complete loss of activity. The data are discussed with respect to our previous observation that bradykinin causes a slow hyperpolarization response in these cell lines and that on prolonged exposure to the peptide the membrane potential response of the cells is lost due to desensitization.
Asunto(s)
Bradiquinina/farmacología , GMP Cíclico/metabolismo , Glioma/metabolismo , Neuroblastoma/metabolismo , Animales , Bradiquinina/análogos & derivados , Línea Celular , Células Híbridas/efectos de los fármacos , Células Híbridas/metabolismo , Cinética , Ratones , Ratas , Relación Estructura-ActividadRESUMEN
A bromodeoxyuridine resistant mutant of rat gliom line C6 grown in the presence of N6,02'-dibutyryl adenosine-3':5'-cyclic monophosphate, although inexcitable by electrical current, responds to iontophoretic application of acetylcholine by slow hyperpolarization. The sensitivity to acetylcholine is strongly reduced by excessive amounts of acetylcholine. The hyperpolarization response is inhibited by atropine or alpha-bungarotoxin, but not by D-tubocurarine. In the absence of acetylcholine some cells display spontaneous hyperpolarizations at regular intervals. The membrane resting potential is decreased if the concentration of external K+ is raised.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Glioma/fisiopatología , Animales , Atropina/farmacología , Bungarotoxinas/farmacología , Línea Celular , Células Cultivadas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Potenciales de la Membrana/efectos de los fármacos , Potasio/farmacología , Ratas , Tubocurarina/farmacologíaRESUMEN
Atrial natriuretic hormones (ANHs) applied to polyploid rat glioma cells induced hyperpolarizations of about 30 s duration, followed by depolarizations lasting 1-2 min. Repeated applications of the peptide resulted in desensitization. The reversal potential of -87 mV at an extracellular K+ concentration of 5 mM and the decrease of membrane resistance during the hyperpolarization indicate that K+ channels were activated by ANH. In these cells the fluorescence signal of 2-[(2-bis[carboxymethyl]amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis [carboxymethyl]aminoquinoline (quin2) was not affected by ANH suggesting that ANH did not change the cytosolic Ca2+-activity.
Asunto(s)
Factor Natriurético Atrial/farmacología , Potenciales de la Membrana/efectos de los fármacos , Tejido Nervioso/efectos de los fármacos , Animales , Bradiquinina/farmacología , Células Cultivadas , Glioma , Células Híbridas , Canales Iónicos/efectos de los fármacos , Neuroblastoma , Potasio/metabolismo , RatasRESUMEN
The function in neurons of the vitamin D-dependent calcium ion-binding-protein of 28 kDa mol. wt., calbindin D28K, is unknown. In order to find a simple system for studying the function of this protein, neuron-rich primary cultures derived from brains of 16-day-old rat embryos were analyzed for the presence of calbindin D28K by immunocytochemical mono- and double-labelling techniques. The studies were carried out between the 5th and 23rd day after seeding. In contrast to the neuronal marker neuron-specific enolase which was found in nearly all cells in the culture, calbindin D28K was expressed only in a subpopulation of neurons. Calbindin D28K-positive cells were intensely stained in their cell bodies and were also stained in their processes. Astroglial cells identified by the presence of the processes. Astroglial cells identified by the presence of the specific marker glial fibrillary acidic protein did not express calbindin D28K. Therefore, calbindin D28K is a useful marker for defining a neuronal subpopulation in neuron-rich primary cultures. Such cultures may be employed as a tool in searching for function(s) of calbindin D28K.