RESUMEN
BACKGROUND AND PURPOSE: Artificial buffers such as HEPES are extensively used to control extracellular pH (pH(e) ) to investigate the effect of H(+) ions on GABA(A) receptor function. EXPERIMENTAL APPROACH: In neurones cultured from spinal cord dorsal horn (DH), dorsal root ganglia (DRG) and cerebellar granule cells (GC) of neonatal rats, we studied the effect of pH(e) on currents induced by GABA(A) receptor agonists, controlling pH(e) with HCO(3) (-) or different concentrations of HEPES. KEY RESULTS: Changing HEPES concentration from 1 to 20 mM at constant pH(e) strongly inhibited the currents induced by submaximal GABA applications, but not those induced by glycine or glutamate, on DH, DRG or GC neurones, increasing twofold the EC(50) for GABA in DH neurones and GC. Submaximal GABA(A) receptor-mediated currents were also inhibited by piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 3-(N-morpholino)propanesulfonic acid, tris(hydroxymethyl)aminomethane or imidazole. PIPES and HEPES, both piperazine derivatives, similarly inhibited GABA(A) receptors, whereas the other buffers had weaker effects and 2-(N-morpholino)ethanesulfonic acid had no effect. HEPES-induced inhibition of submaximal GABA(A) receptor-mediated currents was unaffected by diethylpyrocarbonate, a histidine-modifying reagent. HEPES-induced inhibition of GABA(A) receptors was independent of membrane potential, HCO(3) (-) and intracellular Cl(-) concentration and was not modified by flumazenil, which blocks the benzodiazepine binding site. However, it strongly depended on pH(e) . CONCLUSIONS AND IMPLICATIONS: Inhibition of GABA(A) receptors by HEPES depended on pH(e) , leading to an apparent H(+) -induced inhibition of DH GABA(A) receptors, unrelated to the pH sensitivity of these receptors in both low and physiological buffering conditions, suggesting that protonated HEPES caused this inhibition.
Asunto(s)
Antagonistas de Receptores de GABA-A/farmacología , HEPES/farmacología , Neuronas/efectos de los fármacos , Receptores de GABA-A/metabolismo , Animales , Animales Recién Nacidos , Células CHO , Cerebelo/citología , Cricetinae , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , HEPES/química , Humanos , Concentración de Iones de Hidrógeno , Estructura Molecular , Neuronas/metabolismo , Ratas , Receptores de GABA-A/genética , Médula Espinal/citología , Transmisión Sináptica/efectos de los fármacosRESUMEN
We have previously shown that axonal growth from a subset of sensory neurons was promoted by keratinocytes when the two cell types were co-cultured in a low calcium medium. This phenomenon involves the production of one or several diffusible factors. Here we show that the neuritogenic effect of keratinocytes was significantly reduced in the case of rat primary sensory dorsal root ganglion (DRG) neurons, or completely suppressed in the case of the sensory neuron cell line ND7-23, when the activity of neurotrophin receptors (Trk receptors) was blocked with K252a. This trophic effect apparently involved the activation of tyrosine kinase receptors A and B (TrkA and TrkB) expressed by subpopulations of small- to medium-sized DRG neurons, or only of TrkA receptors in the case of ND7-23 neurons. A residual neurite growth promoting effect of keratinocytes persisted in a fraction of DRG neurons after Trk receptor blockade. This effect was mimicked by the steroid dehydroepiandrosterone (DHEA) but not by other steroids such as pregnenolone, progesterone or 17beta-estradiol. The use of pharmacological agents which inhibit different steps of steroidogenesis indicated that DHEA was probably synthesized from cholesterol in keratinocytes. Our results strongly suggest that DHEA might act as a neurotrophic signal derived from keratinocytes to promote axonal outgrowth from a subpopulation of sensory neurons.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Axones/efectos de los fármacos , Deshidroepiandrosterona/farmacología , Queratinocitos/citología , Factores de Crecimiento Nervioso/farmacología , Células Receptoras Sensoriales/citología , Aminoglutetimida/farmacología , Análisis de Varianza , Animales , Animales Recién Nacidos , Inhibidores de la Aromatasa/farmacología , Axones/fisiología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Carbazoles/farmacología , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Ganglios Espinales/citología , Humanos , Alcaloides Indólicos/farmacología , Queratinocitos/efectos de los fármacos , Neuritas/efectos de los fármacos , Neuritas/fisiología , Ratas , Ratas Wistar , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Factores de TiempoRESUMEN
The pro-apoptotic factor BAX has recently been shown to contribute to Purkinje cell (PC) apoptosis induced by the neurotoxic prion-like protein Doppel (Dpl) in the prion-protein-deficient Ngsk Prnp(0/0) (NP(0/0)) mouse. In view of cellular prion protein (PrP(c)) ability to counteract Dpl neurotoxicity and favor neuronal survival like BCL-2, we investigated the effects of the anti-apoptotic factor BCL-2 on Dpl neurotoxicity by studying the progression of PC death in aging NP(0/0)-Hu-bcl-2 double mutant mice overexpressing human BCL-2 (Hu-bcl-2). Quantitative analysis showed that significantly more PCs survived in NP(0/0)-Hu-bcl-2 double mutants compared with the NP(0/0) mutants. However, number of PCs remained inferior to wild-type levels and to the increased number of PCs observed in Hu-bcl-2 mutants. In the NP(0/0) mutants, Dpl-induced PC death occurred preferentially in the aldolase C-negative parasagittal compartments of the cerebellar cortex. Activation of glial cells exclusively in these compartments, which was abolished by the expression of Hu-bcl-2 in the double mutants, suggested that chronic inflammation is an indirect consequence of Dpl-induced PC death. This partial rescue of NP(0/0) PCs by Hu-bcl-2 expression was similar to that observed in NP(0/0):Bax(-/-) double mutants with bax deletion. Taken together, these data strongly support the involvement of BCL-2 family-dependent apoptotic pathways in Dpl neurotoxicity. The capacity of BCL-2 to compensate PrP(c) deficiency by rescuing PCs from Dpl-induced death suggests that the BCL-2-like property of PrP(c) may impair Dpl-like neurotoxic pathways in wild-type neurons.
Asunto(s)
Apoptosis/efectos de los fármacos , Priones/genética , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Células de Purkinje/efectos de los fármacos , Factores de Edad , Análisis de Varianza , Animales , Recuento de Células , Cerebelo/citología , Fructosa-Bifosfato Aldolasa/metabolismo , Proteínas Ligadas a GPI , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Priones/toxicidadRESUMEN
Research efforts to deduce the function of the prion protein (PrPc) in knock-out mouse mutants have revealed that large deletions in the PrPc genome result in the ectopic neuronal expression of the prion-like protein Doppel (Dpl). In our analysis of one such line of mutant mice, Ngsk Prnp0/0 (NP0/0), we demonstrate that the ectopic expression of Dpl in brain neurons induces significant levels of cerebellar Purkinje cell (PC) death as early as six months after birth. To investigate the involvement of the mitochondrial proapoptotic factor BAX in the Dpl-induced apoptosis of PCs, we have analyzed the progression of PC death in aging NP0/0:Bax-/- double knockout mutants. Quantitative analysis of cell numbers showed that significantly more PCs survived in NP0/0:Bax-/- double mutants than in the NP0/0:Bax+/+ mutants. However, PC numbers were not restored to wildtype levels or to the increased number of PCs observed in Bax-/- mutants. The partial rescue of NP0/0 PCs suggests that the ectopic expression of Dpl induces both BAX-dependent and BAX-independent pathways of cell death. The activation of glial cells that is shown to be associated topographically with Dpl-induced PC death in the NP0/0:Bax+/+ mutants is abolished by the loss of Bax expression in the double mutant mice, suggesting that chronic inflammation is an indirect consequence of Dpl-induced PC death.
Asunto(s)
Apoptosis/fisiología , Priones/fisiología , Células de Purkinje/fisiología , Proteína X Asociada a bcl-2/fisiología , Animales , Proteínas de Unión al Calcio/metabolismo , Recuento de Células , Femenino , Técnica del Anticuerpo Fluorescente , Proteínas Ligadas a GPI , Genotipo , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/patología , Procesamiento de Imagen Asistido por Computador , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Priones/genética , Priones/metabolismo , Células de Purkinje/metabolismo , Proteína X Asociada a bcl-2/genéticaRESUMEN
In Xenopus oocytes, metaphase II arrest is due to a cytostatic factor (CSF) that involves c-Mos, maintaining a high MPF (cdk1/cyclin B) activity in the cell. At fertilization, a rise in intracellular calcium triggers the proteolysis of both cyclin B and c-Mos. The kinase inhibitor 6-dimethylaminopurine (6-DMAP) is also able to release matured Xenopus oocytes from metaphase II block. This is characterized by c-Mos proteolysis without degradation of cyclin B. We hypothesized that 6-DMAP induced an increase in intracellular calcium. Using the calcium-sensitive fluorescent dye Fura-2, we observed a systematic increase in intracellular calcium following 6-DMAP application. In matured oocytes previously microinjected with the calcium chelator BAPTA, no calcium changes occurred after 6-DMAP addition; however, c-Mos was still proteolysed. In oocytes at the GVBD stage, c-Mos proteolysis occurred in response to 6-DMAP but not to calcium ionophore treatment. We suggest that c-Mos proteolysis is rather controlled by a phosphorylation-dependent process.
Asunto(s)
Adenina/análogos & derivados , Adenina/farmacología , Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Oocitos/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas Proto-Oncogénicas c-mos/metabolismo , Xenopus laevis/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Femenino , Colorantes Fluorescentes/farmacocinética , Fura-2/farmacocinética , Metafase/efectos de los fármacos , Metafase/fisiología , Oocitos/citología , Oocitos/efectos de los fármacos , Péptido Hidrolasas/efectos de los fármacos , Péptido Hidrolasas/genética , Proteínas Proto-Oncogénicas c-mos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mos/genética , Xenopus laevis/embriologíaRESUMEN
Intracellular pH was measured with the pH-sensitive fluorescent probe BCECF in spinal cord neurones cultured from rat embryos. At an external pH of 7.3, the average steady-state pHi was 7.18 +/- 0.03 (SEM, n = 97) and 7.02 +/- 0.01 (n = 221) in HEPES-buffered and in bicarbonate-buffered medium, respectively. In both external media, pHi was strongly dependent on external pH (pHe). In HEPES-buffered medium, pHi recovery following an acid load induced by transient application of ammonium required external Na+ and was inhibited by amiloride, indicating the presence of a Na+/H+ exchange. Na(+)- and HCO3(-)-dependent, DIDS-sensitive alkalinizing mechanisms also contributed to pHi regulation in CO2/bicarbonate-buffered medium. The presence of an electrogenic Na(+)-HCO3- cotransporter was confirmed by the alkalinizing effect of KCl application. The fact that pHi is lower in CO2/bicarbonate- than in HEPES-buffered medium and the alkalinization observed upon suppression of external Cl- suggest that the acidifying Cl-/HCO3- transporter plays an important role in defining pHi.
Asunto(s)
Equilibrio Ácido-Base/fisiología , Células del Asta Anterior/metabolismo , Antiportadores/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Amilorida/farmacología , Cloruro de Amonio/farmacología , Animales , Células del Asta Anterior/embriología , Tampones (Química) , Líquido Cefalorraquídeo/química , Medios de Cultivo/química , Embrión de Mamíferos/metabolismo , Fluoresceínas/farmacología , Colorantes Fluorescentes/farmacología , Concentración de Iones de Hidrógeno , Ratas , Bicarbonato de Sodio/metabolismoRESUMEN
The pineal gland is mainly innervated by sympathetic fibres containing noradrenaline (NA) and neuropeptide Y (NPY). NA released at night stimulates melatonin synthesis via a beta1-adrenergic-induced increase in cyclic adenosine monophosphate (cAMP) concentration potentiated by an alpha1-adrenergic-induced increase in Ca2+ concentration. We previously showed that NPY acted on presynaptic Y2 receptors inhibiting NA release and on postsynaptic Y1 receptors stimulating melatonin synthesis. Here we used Fura-2 imaging to assess the effect of NPY on the intracellular Ca2+ concentration, [Ca2+]i, in cultured rat pineal cells. In 84% of cells, on average, 10 nM NPY induced a progressive rise of [Ca2+]i from its basal value of 102+/-3 nM to a plateau of 180+/-6 nM (n = 467 cells), which lasted the time of NPY application. This effect of NPY appeared dependent on extracellular Ca2+.
Asunto(s)
Calcio/metabolismo , Neuropéptido Y/farmacología , Glándula Pineal/metabolismo , Fibras Adrenérgicas/metabolismo , Animales , Calcio/farmacocinética , Células Cultivadas , Colorantes Fluorescentes , Fura-2 , Glándula Pineal/citología , Glándula Pineal/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
We have investigated, in mice, an in vivo method for producing low-lactose milk, based on the creation of transgenic animals carrying a hybrid gene in which the intestinal lactase-phlorizin hydrolase cDNA was placed under the control of the mammary-specific alpha-lactalbumin promoter. Transgenic females expressed lactase protein and activity during lactation at the apical side of mammary alveolar cells. Active lactase was also secreted into milk, anchored in the outer membrane of fat globules. Lactase synthesis in the mammary gland caused a significant decrease in milk lactose (50-85%) without obvious changes in fat and protein concentrations. Sucklings nourished with low-lactose milk developed normally. Hence, these data validate the use of transgenic animals expressing lactase in the mammary gland to produce low-lactose milk in vivo, and they demonstrate that the secretion of an intestinal digestive enzyme into milk can selectively modify its composition.
Asunto(s)
Intestinos/enzimología , Lactosa/análisis , Glándulas Mamarias Animales/enzimología , Leche/química , beta-Galactosidasa/genética , Animales , Secuencia de Bases , Western Blotting , Cromatografía en Capa Delgada , ADN Complementario , Femenino , Expresión Génica , Lactasa , Intolerancia a la Lactosa , Ratones , Ratones Transgénicos , beta-Galactosidasa/biosíntesisRESUMEN
The effect of the local anaesthetic procaine on the intracellular pH, pHi, and electrophysiological properties of full-grown Xenopus oocytes was studied. In spite of its interference with both the pH-sensitive microelectrodes and fluorescent probe BCECF, we have shown that procaine induced an intracellular acidification rather than the alkalization commonly observed in most cells. The resting pHi of Xenopus oocytes loaded with BCECF was 7.36 +/- 0.04 (n = 16). Addition of 10 mM procaine to the bath at pH 7.5 caused pHi to decrease to a new steady state value of 6.97 +/- 0.05 (n = 9). A similar behaviour of pHi was observed with microelectrodes. Procaine also promoted a rise in membrane conductance and a membrane depolarization. These changes in membrane potential and conductance were not caused by the decrease in pHi since the addition of sodium propionate at pH 7.5 produced the same decrease of pHi as procaine, but resulted in only a slight depolarization with superimposed oscillations. Current measurements using two-electrode voltage clamp showed that the depolarization was associated with an inward current. No significant effect on this current was observed when replacing Cl, K or Na in the external medium. The absence of effect of Cl and K channel inhibitors argues against the involvement of Cl and K currents during the procaine response.
Asunto(s)
Antimetabolitos/farmacología , Oocitos/efectos de los fármacos , Procaína/farmacología , Cloruro de Amonio/farmacología , Animales , Femenino , Concentración de Iones de Hidrógeno , Líquido Intracelular , Potenciales de la Membrana/efectos de los fármacos , Oocitos/fisiología , Xenopus laevisRESUMEN
The neuropeptide substance P (SP) is known to play a key role in peripheral nociceptive processes. We investigated the in vitro pharmacological characteristics of functional tachykinin receptors expressed in dorsal root ganglia (DRG) sensory neurones by analysing intracellular free calcium concentration changes induced after stimulation by SP or specific tachykinin agonists. We observed that about 37% of the tested neurones were responsive to either SP or an NK1-, NK2- or NK3-specific agonist. Tachykinin-responsive neurones had a small soma diameter (<20 microm) and were sensitive to capsaicin. These results suggest the presence of NK1, NK2 and NK3 receptors in noxious sensory neurones.
Asunto(s)
Ganglios Espinales/química , Neuronas Aferentes/química , Receptores de Taquicininas/análisis , Análisis de Varianza , Animales , Células Cultivadas , Ganglios Espinales/citología , Neuroquinina A/farmacología , Neuroquinina B/farmacología , Ratas , Ratas Wistar , Receptores de Taquicininas/agonistas , Sustancia P/farmacologíaRESUMEN
When a high density of alpha1-subunit glycine receptor (GlyR) is expressed in Xenopus oocytes, two populations of channels can be distinguished according to their apparent affinity for glycine which differs 5- to 6-fold. To compare the open pore diameter of these channels, the relative permeability of formate with respect to chloride (P(formate)/P(Cl)) was determined in bionic conditions. For the low-affinity GlyR P(formate)/P(Cl) was comparable to that reported for glycine-gated channels in cultured spinal cord and hippocampal neurons. In contrast, the high-affinity GlyR had a 56% larger P(formate)/P(Cl). In addition, the open probability of the channels was differentially sensitive to voltage. These results show that the high expression of alpha1 GlyR resulted in two populations of GlyR which differed not only in the affinity to agonists but also in permeation and gating mechanisms.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Glicina/metabolismo , Activación del Canal Iónico/fisiología , Receptores de Glicina/metabolismo , Animales , Cloruros/metabolismo , ADN Complementario , Electrofisiología , Formiatos/metabolismo , Microinyecciones , Oocitos , Técnicas de Placa-Clamp , Receptores de Glicina/química , Transfección , XenopusRESUMEN
The survival of rat cerebellar granule cells maintained in vitro is enhanced by a KCl-enriched medium. This effect is classically interpreted as resulting from a higher cytosolic calcium concentration. This implies the presence of voltage-dependent Ca2+ channels and a membrane potential that can respond to changes in external K+. Since previous studies cast a doubt on these two conditions, we reinvestigated the resting membrane potential and Ca2+ influxes in rat cerebellar granule neurones during the first week in vitro using a fluorescence imaging approach. Membrane potential was assessed with the fluorescent dye bis-oxonol, and intracellular free calcium with Fura-2. Resting potential was shown to progressively decrease from -40 mV at the first day in vitro to -60 mV at day 7. At all times in culture, as early as day 0, cells were depolarized when external KCl concentration was increased from 5 to 30 mM. This depolarization resulted in an increased cytosolic calcium concentration due to Ca2+ influx through L-type and N-type voltage-activated Ca2+ channels, functional at day 0. Gross estimations of the permeabilities of Na+ and Cl- were obtained at various times in culture by measuring the changes in resting potential brought about by a reduction of their external concentration. A progressive increase of the relative permeability to K+ ions seems to underlie the evolution of the resting potential with time.
Asunto(s)
Cerebelo/citología , Animales , Calcio/metabolismo , Canales de Calcio/fisiología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Cloruros/metabolismo , Colorantes Fluorescentes , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Potasio/metabolismo , Cloruro de Potasio/farmacología , Ratas , Ratas Wistar , Sodio/metabolismo , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , TiobarbitúricosRESUMEN
Forskolin, routinely used as a specific activator of the cAMP pathway, is also a blocker of various ionic channels in a cAMP-independent way. We investigated, in rat cerebellar granule cells in culture, the effects of forskolin and its structural analogue 1,9-dideoxyforskolin on Ca2+ entry. Changes in cytosolic free Ca2+ concentration ([Ca]i) were monitored using fura-2 microfluorimetry. The increase in [Ca]i observed in response to membrane depolarization by 30 mM KCI was reduced by 20% in the presence of 100 microM forskolin, and by 71% with the same concentration of 1,9-dideoxyforskolin. A dose-response curve for 1,9-dideoxyforskolin gave an estimated IC50 of 54 microM. Additional experiments using the patch-clamp technique showed that 100 microM 1,9-dideoxyforskolin inhibit voltage-activated Ca2+ currents by 63%, although forskolin had no significant effect in the same conditions. This blocking effect of 1,9-dideoxyforskolin is not specific of a given Ca2+ channel type.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Colforsina/análogos & derivados , Animales , Bario/metabolismo , Células Cultivadas , Colforsina/farmacología , Relación Dosis-Respuesta a Droga , Fura-2 , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , RatasRESUMEN
In Xenopus laevis oocytes a transient increase in intracellular pH has been reported to occur during progesterone-induced maturation. Using a cytological approach, we have systematically analyzed germinal vesicle breakdown and meiotic spindle formation in various experimental conditions either preventing or promoting pHi changes. Injection of a neutral buffer (MOPS pH 6.9) induced a cytosolic acidification of 0.3 pH unit and inhibited by 30% the formation of the maturation white spot after progesterone exposure; in oocytes displaying a white spot, only half showed a spindle, often located far from the plasma membrane. Similar results were observed with a Na-free medium which prevents oocyte alkalization. Injection of an alkaline buffer (Tris pH 9) was able to induce migration of the germinal vesicle in 25% of the oocytes in the absence of progesterone, but failed to induce GVBD. Taken together, these results suggest that the increase in pHi observed during maturation may be involved in the migration of the germinal vesicle towards the plasma membrane. We also incubated oocytes in the presence of procaine, a weak base often used to artificially alkalize the oocyte cytoplasm. The changes induced by exposure to procaine were different from those resulting from alkaline buffer injection. Indeed procaine promoted GVBD, as well as spindle formation and chromosome condensation. However these events appeared without migration of the germinal vesicle, suggesting that the expected alkalization did not occur.
Asunto(s)
Equilibrio Ácido-Base/fisiología , Citoplasma/fisiología , Oocitos/crecimiento & desarrollo , Xenopus laevis/fisiología , Equilibrio Ácido-Base/efectos de los fármacos , Animales , Tampones (Química) , Núcleo Celular/metabolismo , Medios de Cultivo , Femenino , Técnicas In Vitro , Morfolinas/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Procaína/farmacología , Progesterona/farmacología , Profase/fisiología , Sodio/farmacologíaRESUMEN
Acute injection of haloperidol, a dopamine D2 receptor antagonist, is known to increase immediate early gene expression of the fos and jun families in rodent striatal neurons. A set of gene induction, including c-fos, jun B and TIS8/egr-1, was found when haloperidol was added to PC12 cells in culture. Electrophoretic mobility-shift assays show that haloperidol-evoked gene induction was accompanied by a transient and dose-dependent increase in AP1 and EGR-1 binding activities in these cells. Gene expression is tentatively explained by the rapid and transient increase in cytosolic free Ca2+ concentration observed upon haloperidol addition. The cytosolic calcium rise and AP1 binding activation elicited by haloperidol were dependent on extracellular Ca2+, suggesting that haloperidol exerted its effects by promoting Ca2+ entry into PC12 cells. The haloperidol-induced increase in AP1 binding activity and intracellular Ca2+ was not reproduced by two other dopamine D2 receptor antagonists, sulpiride and (+)-butaclamol.
Asunto(s)
Calcio/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes fos , Genes jun , Haloperidol/farmacología , Animales , Secuencia de Bases , Citosol/efectos de los fármacos , Citosol/metabolismo , Datos de Secuencia Molecular , Células PC12 , Ratas , Ratas Wistar , Transcripción Genética/efectos de los fármacos , Activación TranscripcionalRESUMEN
Oxygen consumption rate (MO2) of hen eggs was measured on incubation day 16 (37.8 degrees C, 55% humidity) during acute exposure (90 min) to ambient hyperoxia (FI02 = 0.42) or hypoxia (FIO2 = 0.105). During the last part of these exposures, an H2 washout method was used to estimate relative changes in chorioallantoic membrane (CAM) blood flow, taking as an index the net change in the H2 washout rate constant between any experimental condition and the circulation arrested egg. Doubling normoxic FIO2 increased MO2 to an asymptotic value which was 4% above the normal (P less than 0.05; MO2 in normoxia = 890 mumols/h) even after correcting for the normoxic increase in MO2 with time during development (delta MO2/delta t = 21.5 mumols/h2; P less than 0.001). Halving FIO2 reduced MO2 calculated in the same way to 388 mumols/h. The estimate of the CAM blood flow, relative to normoxia, was 1.12 in hyperoxia (not significant, P = 0.05) and 0.68 in hypoxia (P less than 0.001). The limited changes in CAM blood flow and MO2 during hyperoxia indicate that they are both already close to their maximal values in normoxia. During acute hypoxia the 16 day embryo behaves as an oxygen-conformer; however, the small relative decrease in MO2 per unit of the flow index observed during hyperoxia suggests that the embryo can regulate its CAM blood flow to a small extent. The survival of the embryo and its recovery from hypoxia without a detectable O2 repayment suggest small if any anaerobic regulatory pathways and indicate a true metabolic depression.
Asunto(s)
Alantoides/irrigación sanguínea , Corion/irrigación sanguínea , Consumo de Oxígeno , Alantoides/metabolismo , Animales , Embrión de Pollo , Corion/metabolismo , Hidrógeno/metabolismo , Hipoxia , Cinética , Estadística como AsuntoRESUMEN
The hanging drop method for protein crystallization consists of equilibrating a water droplet containing the protein and a precipitant against a reservoir solution at a higher precipitant concentration. The time for water equilibration--which affects the kinetics of crystallization--to reach 90% of completion is shown to vary between about 25 h and more than 25 days, depending on experimental conditions. Experiments were performed with three of the most widely used precipitants (ammonium sulfate, polyethylene glycol, 2-methyl-2,4-pentanediol), combining various parameters expected to affect the rate of water evaporation. The most dramatic effects were obtained when varying temperature and initial drop volume. A simple empirical equation for estimating the kinetics of water equilibration under given crystallization conditions is proposed.
Asunto(s)
Cristalización , Proteínas , Agua , Fenómenos Químicos , Precipitación Química , Química Física , Cinética , Modelos Químicos , MuramidasaRESUMEN
Dissociated capillary endothelial cells display a voltage-dependent Ca current activating around the resting potential. The initial transient component of the current corresponds to a Ca channel of the T type. Some cells also display a plateau component corresponding to a distinct dihydropyridine-sensitive Ca channel. Depolarization induced by high external K+ elicits an increase in cytoplasmic Ca concentration. Confluent cells have been found to express the same Ca permeabilities.
Asunto(s)
Canales de Calcio/fisiología , Endotelio Vascular/fisiología , Médula Suprarrenal/fisiología , Amilorida/farmacología , Animales , Calcio/metabolismo , Capilares/fisiología , Bovinos , Células Cultivadas , Citosol/metabolismo , Conductividad Eléctrica , Electrofisiología/métodos , Potenciales de la Membrana/efectos de los fármacosRESUMEN
The patch-clamp technique in whole-cell configuration was used to investigate the kinetics of decay of calcium currents in rat sensory neurones. Whole-cell recording permitted control of the internal medium, particularly of the internal free calcium concentration, which was maintained at either 10(-9) M or 10(-6) M using a high concentration of Ca buffer. The inactivation decay of the total Ca current elicited above -10 mV was found to be faster at pCa 6 than at pCa 9. The total current contained three exponential components which were tentatively identified as the three types of Ca currents (IcaT, IcaN and IcaS). Kinetic analyses indicated that the control of the inactivation process by internal Ca results from an effect on both high-threshold Ca currents, IcaN and IcaS. The inactivation kinetics reported in the literature presents a large variability depending on the cell type. We propose that this variability may result from differences in the capacity of those cells to control their internal Ca.
Asunto(s)
Canales de Calcio/fisiología , Neuronas Aferentes/fisiología , Animales , Células Cultivadas , Electrofisiología , Ganglio Nudoso/citología , RatasRESUMEN
Intracellular pH was determined (DMO method) in European hamsters, in the spontaneously-occurring respiratory acidosis of hibernation, in hypercapnia due to breathing 12% CO2 in air in euthermy in spring, and in euthermicnormocapnic controls. From euthermy to hibernation, the temperature coefficient of pH was lowest in blood plasma and brain, intermediate in striated muscles (thigh muscles and diaphragm), and highest in heart and liver (Fig. 1). Correspondingly, the estimated dissociation ratio of the protein imidazole buffer groups, alpha Im, decreased markedly in plasma and brain, denoting an acid titration, but varied little in liver and heart. Striated muscles were intermediate (Fig. 2). Like in other mammals, intracellular responses to short-term euthermic respiratory acidosis were characterized by a partial metabolic compensation in the brain and a small metabolic acidification in striated muscles. In hibernation, a powerful metabolic compensation took place in liver and heart, nearly restoring alpha Im, but none occurred in brain (Figs. 3 to 5). The existence of an intracellular acidosis in brain and striated muscles during hibernation is in keeping with an inhibitory role of acidosis, whereas the homeostasis of intracellular alpha Im in liver and heart would subserve the eurythermal functioning of metabolic regulations in these organs, like in most organs of ectotherms.