RESUMEN
Nesfatin-1 is a recently identified 82 amino acid peptide shown to have an anorexigenic effect on rodents when administrered centrally and peripherally. Nesfatin-1 is expressed not only in neurones of various brain areas, including the hypothalamic and brainstem nuclei, but also in peripheral organs, such as the stomach and the pancreas. Nesfatinergic neurones were reported to participate in the regulation of satiety signals and in the responses to other stimuli, including restraint stress, abdominal surgery, and lipopolysaccharide-induced inflammation. The present study aimed to investigate whether NUCB2/nesfatin-1 expressing neurones also take part in the central signalling activated in response to hypoglycaemia and therefore are involved in central glucose sensing. Using immunolabelling methods based on the detection of the neuronal activation marker c-Fos and of nesfatin-1, we showed that peripheral injection of insulin induced a strong activation of nesfatin-1-expressing neurones in the brain vagal-regulatory nuclei, including the arcuate nucleus, paraventricular nucleus, lateral hypothalamic area, dorsal motor nucleus of the vagus (DMNX) and nucleus of the tractus solitarius. In response to intracellular glucopaenia induced by i.p. or i.c.v. 2-deoxyglucose injection, the c-Fos/nesfatin-1 colocalisations observed at the hypothalamic and brainstem levels were similar to those observed after insulin-induced hypoglycaemia. Moreover, using Fluorogold as a retrograde tracer, we showed that nesfatinergic preganglionic DMNX neurones activated by hypoglycaemia target the stomach and the pancreas. Taken together, these results suggest that a subpopulation of nesfatinergic neurones belongs to the central network activated by hypoglycaemia, and that nesfatin-1 participates in the triggering of physiological and hormonal counter-regulations observed in response to hypoglycaemia.
Asunto(s)
Tronco Encefálico/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al ADN/metabolismo , Hipoglucemia/metabolismo , Hipotálamo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Animales , Hipoglucemia/inducido químicamente , Insulina , Masculino , Vías Nerviosas/metabolismo , Nucleobindinas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas WistarRESUMEN
Whole-cell patch clamp recordings were performed on neurones in the lamina X of rat spinal cord slices in order to characterize glycinergic synaptic currents and their modulation by nicotinic acetylcholine receptors. In the presence of TTX, bicuculline and kynurenic acid, glycine-induced currents and miniature glycinergic postsynaptic currents (mIPSCs) were recorded. These currents reversed near the chloride ion equilibrium potential and were blocked by strychnine (1 microM). A selective nicotinic acetylcholine receptor (nAChR) agonist 1,1-dimethyl-4-phenyl-piperazinium (DMPP), increased the frequency of glycinergic mIPSCs without altering significantly their amplitude distributions or their kinetic properties. The effects of DMPP were mimicked by different nAChRs agonists with the following apparent order of potency: ACh > DMPP > nicotine > cytisine. The effect of DMPP on mIPSCs was blocked by both d-tubocurarine and hexamethonium, and was reduced by dihydro-beta-erythroidine and methyllycaconitine (MLA), antagonists of non alpha7- and alpha7-containing nAChRs, respectively. In the absence of TTX, strychnine-sensitive glycinergic electrically evoked postsynaptic currents (eIPSCs) could be recorded. DMPP blocked the appearance of electrically evoked IPSCs while still inducing the appearance of spontaneous glycine IPSCs. These data demonstrate that neurones surrounding the central canal of the spinal cord present a glycinergic synaptic transmission which is modulated by terminal nAChRs.
Asunto(s)
Aconitina/análogos & derivados , Glicina/metabolismo , Potenciales de la Membrana , Neuronas/metabolismo , Receptores Nicotínicos/fisiología , Médula Espinal/citología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Aconitina/farmacología , Alcaloides/farmacología , Anestésicos Locales/farmacología , Animales , Animales Recién Nacidos , Azocinas , Bicuculina/farmacología , Dihidro-beta-Eritroidina/farmacología , Yoduro de Dimetilfenilpiperazina/farmacología , Interacciones Farmacológicas , Antagonistas de Aminoácidos Excitadores/farmacología , Glicinérgicos/farmacología , Hexametonio/farmacología , Técnicas In Vitro , Ácido Quinurénico/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp/métodos , Quinolizinas , Ratas , Ratas Wistar , Médula Espinal/fisiología , Estricnina/farmacología , Tetrodotoxina/farmacología , Factores de Tiempo , Tubocurarina/farmacologíaRESUMEN
Using patch clamp recordings on neonatal rat spinal cord slices, we have looked for the presence of alpha-bungarotoxin-sensitive nicotinic ACh receptors (nAChRs) on sympathetic preganglionic neurones (SPNs) surrounding the central canal of the spinal cord (lamina X) and examined whether they were implicated in a fast cholinergic synaptic transmission. SPNs were identified either by their morphology using biocytin in the recording electrode and/or by antidromic stimulation of the ventral rootlets. The selective alpha7-containing nAChR (alpha7*nAChR) agonist choline (10 mM) induced a fast, rapidly desensitizing inward current, which was fully blocked by alpha-bungarotoxin (alpha-BgT; 50 nM) and strychnine (1 microM), two antagonists of alpha7*nAChRs. The I-V relationship of the choline-induced current showed a strong inward-going rectification. Electrically evoked excitatory postsynaptic currents (eEPSCs) could be recorded. At -60 mV, eEPSCs peaked at -26.2 pA and decayed monoexponentially with a mean time constant of 8.5 ms. The current-voltage relationship for eEPSCs exhibited a strong inward rectification and a reversal potential close to 0 mV, compatible with a non-selective cationic current. The appearance of eEPSCs was entirely suppressed by the application of 100 microM ACh or nicotine. Choline (10 mM) and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP; 100 microM) both reduced the amplitude of eEPSCs, whereas cytisine (100 microM) had no effect. Strychnine (1 microM) and alpha-BgT (50 nM) both suppressed the eEPSCs. Blocking the P2X purinergic and 5-HT(3) receptors had no effect on eEPSCs. DMPP induced four types of current, which differed in their onset and desensitization rate. The most frequently encountered responses were insensitive to the action of strychnine and alpha-BgT, and were reproduced by ACh and nicotine but not by cytisine. We conclude that SPNs of the lamina X express several classes of nAChRs and in particular alpha-BgT-sensitive nAChRs. This is the first demonstration in a mammalian spinal cord preparation of a fast cholinergic neurotransmission in which alpha-BgT-sensitive nicotinic receptors are involved.
Asunto(s)
Bungarotoxinas/farmacología , Neuronas/fisiología , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/fisiología , Médula Espinal/fisiología , Transmisión Sináptica/fisiología , Animales , Colina/farmacología , Yoduro de Dimetilfenilpiperazina/farmacología , Conductividad Eléctrica , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Técnicas In Vitro , Neuronas/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Subunidades de Proteína , Ratas , Ratas Wistar , Tiempo de Reacción , Médula Espinal/efectos de los fármacos , Estricnina/farmacología , Sinapsis/fisiologíaRESUMEN
Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca2+]i) associated with periodic Ca2+ oscillations during prolonged ACh applications. The early transient rise in [Ca2+]i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca2+ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca2+]i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca2+-sensitive K+ currents. These K+ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca2+ from intracellular stores which in turn activate Ca2+-dependent (BK-type) K+ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Muscarina/metabolismo , Agonistas Muscarínicos/metabolismo , Canales de Potasio Calcio-Activados , Canales de Potasio/metabolismo , Acetilcolina/farmacología , Astrocitoma , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio , Muscarina/farmacología , Agonistas Muscarínicos/farmacología , Células Tumorales CultivadasRESUMEN
1. Nicotinic responses and actions on excitatory synaptic activity were studied in eighty-four neurones in the region dorsal to the central canal (lamina X) in transverse thoracolumbar spinal cord slices of neonate (P2-P10) rats by using the whole-cell patch-clamp technique. 2. Neurones (n = 15) labelled with Lucifer Yellow, showed the typical morphology of sympathetic preganglionic neurones (SPNs) in the central autonomic area (CA). Unlabelled neurones of comparable morphology were visually identified and recorded. 3. All neurones recorded responded to the nicotinic acetylcholine receptor (nAChR) agonist, DMPP. Under current-clamp conditions, pressure ejections of DMPP depolarized cells and induced the discharge of action potentials. Tetrodotoxin suppressed action potentials but not DMPP-induced depolarization. 4. Under voltage-clamp conditions at a holding potential (Vh) of -50 mV, DMPP induced a transient inward current (which reversed around 0 mV) and an increase in membrane current noise in 50% of the recorded neurones. In the others, DMPP increased membrane current noise without measurable inward current. The current-voltage relationship showed strong inward rectification at holding potentials more positive than 0 mV. 5. In neurones displaying a detectable current response to DMPP, the following agonist rank order potency could be established: DMPP = nicotine > cytisine > ACh. The DMPP response could be blocked by mecamylamine but was insensitive to methyllycaconite. 6. Pressure application of glutamate induced inward currents in all cells tested at a Vh of -50 mV. This response reversed at 10 mV, displayed a region of negative slope conductance at Vh more negative than -30 mV and was partially blocked by CNQX. Pressure application of DMPP transiently increased the amplitude of the glutamate-induced current in six out of nine cells tested. This potentiation persisted in the presence of tetrodotoxin. 7. Forty per cent of the recorded neurones displayed spontaneous excitatory postsynaptic currents (sEPSCs). At a Vh of -50 mV the sEPSCs had a mean amplitude of -19.3 pA and occurred at a frequency below 0.5 Hz. sEPSCs were blocked by CNQX and inverted around 0 mV. Brief application of DMPP increased the discharge frequency of sEPSCs without affecting their kinetics. Additionally, in some cells DMPP increased mean sEPSC amplitude. 8. Focal electrically evoked EPSCs reversed close to 10 mV and were sensitive to CNQX. They occurred with a constant latency, rise time and a mono-exponential decay time. Application of DMPP decreased the percentage of stimulation failures and increased the amplitude of evoked EPSCs, in all cells tested. 9. It is concluded that neurones in the CA, presumed to be SPNs, have functional nAChRs with activation having two distinct effects: firstly, a direct depolarization of the postsynaptic membrane; and secondly, a facilitation of the excitatory transmission onto these cells. This second effect is achieved by an increase of the size of the glutamate-induced current at the postsynaptic level as well as by an enhancement of the presynaptic release of glutamate.
Asunto(s)
Agonistas Colinérgicos/farmacología , Receptores Colinérgicos/metabolismo , Médula Espinal/metabolismo , Transmisión Sináptica/efectos de los fármacos , Acetilcolina/farmacología , Alcaloides/farmacología , Animales , Azocinas , Yoduro de Dimetilfenilpiperazina/farmacología , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Ácido Glutámico/farmacología , Isoquinolinas/metabolismo , Mecamilamina/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Nicotina/farmacología , Técnicas de Placa-Clamp , Quinolizinas , Ratas , Ratas Wistar , Tetrodotoxina/farmacologíaRESUMEN
1. Nicotinic acetylcholine receptors (nAChRs) on lamina X neurones in neonate (P1-P12) rat transverse thoracolumbar spinal cord slices were studied using the whole-cell patch-clamp technique. These visually selected neurones are located dorsal to the central canal, mainly in the ventral half of the dorsal commissure. 2. Pressure application of the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium (DMPP) (1 mM) induced a rapid depolarization on which action potentials are superimposed. 3. At -50 mV, DMPP (1 mM), pressure ejected for 100 ms, induced a fast inward current with a mean amplitude of -280 pA (n = 28) in 90% of the neurones recorded. Superfusion of tetrodotoxin (TTX), a solution containing 0 Ca(2+)-high Mg2+, CdCl2 or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) did not abolish the DMPP-induced current, which confirmed a direct postsynaptic effect of DMPP on recorded neurones. 3. The current-voltage (I-V) relationship for DMPP-induced current exhibited a reversal potential of 0 mV (NaCl outside, potassium gluconate inside) and a strong inward rectification. 4. The DMPP-induced responses were blocked by mecamylamine, hexamethonium and d-tubocurarine (dTC) but were insensitive to alpha-bungarotoxin and methyllycaconitine (MLA). 5. We conclude that lamina X neurones located dorsally to the central canal possess nicotinic acetylcholine receptors. Activation of these nicotinic receptors results in depolarization and generation of action potentials. These receptors may be involved in the modulation of the somato- and viscerosensory transmission.
Asunto(s)
Potenciales de la Membrana/efectos de los fármacos , Neuronas/fisiología , Receptores Nicotínicos/fisiología , Médula Espinal/fisiología , Animales , Yoduro de Dimetilfenilpiperazina/farmacología , Hexametonio/farmacología , Técnicas de Placa-Clamp , Ratas , Factores de TiempoRESUMEN
Activation of both calcium and AMP-dependent regulatory pathways promotes survival of cerebellar neurons in vitro. Complex cellular programs such as survival must involve precise genetic responses. We show here, at the genomic level, that depolarization potentiates AMP-driven transcription of a variety of genes including the c-fos and c-jun proto-oncogenes, and the gene for somatostatin, proenkephalin and nerve growth factor. We used a reporter gene driven by the minimal AMP-responsive element (TGACGTCA) as a model system for studying this class of genes. In primary neurons, this reporter construct is co-activated in a synergistic manner by forskolin and KCl. We show that, in contrast to AMP, calcium-driven transcription does not require functional AMP-dependent protein kinase. Thus, when calcium and AMP levels are increased, these two second messengers stimulate transcription through different kinases which converge at the level of the AMP-responsive element. In addition, lower levels of intracellular free calcium can potentiate AMP-dependent transcription. This effect results from increased cyclic AMP accumulation and is strictly mediated by the AMP/AMP-dependent protein kinase pathway. In summary, low and high calcium concentrations potentiate AMP-dependent transcription via distinct mechanisms. Low calcium increases AMP production, whereas high calcium activates a non-cyclic AMP-dependent protein kinase, which in turn synergizes with AMP-activated transcription. These distinct mechanisms are likely to operate under specific physiological conditions within the neuronal network.
Asunto(s)
Calcio/metabolismo , Calcio/farmacología , AMP Cíclico/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colforsina/farmacología , Proteínas Quinasas/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Transient outward currents (A-currents; IA) were recorded in sympathetic preganglionic neurones (SPNs) and glial cells of the intermediolateral cell column (IML) by whole-cell recordings in rat spinal cord slices. In both cell types IA activated at around -45 mV and the time-course of decay was monoexponential, but faster in glial cells than in neurones. In both cases decay time constants displayed the same increase with depolarization above -30 mV. In neurones, the activation curve was shifted to more negative value along the voltage axis and was steeper than the activation curve for glial cells whereas inactivation curves were similar. Recovery from inactivation followed a double and monoexponential decay in neurones and glial cells, respectively.
Asunto(s)
Fibras Autónomas Preganglionares/citología , Neuroglía/fisiología , Canales de Potasio/fisiología , Médula Espinal/citología , Sistema Nervioso Simpático/citología , Animales , Animales Recién Nacidos , Técnicas In Vitro , Cinética , Región Lumbosacra , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
Using perforated patch recordings in combination with intracellular Ca2+ ([Ca2+]i) fluorescence measurements, we have identified a delayed Ca(2+)-dependent Cl- current in a mammalian sympathetic ganglion cell. This Cl- current is induced by the synergistic action of Ca2+ and diacylglycerol (DAG) and is blocked by inhibitors of protein kinase C. As a result, the current can be induced by acetylcholine through the conjoint activation of nicotinic receptors (to produce a rise in [Ca2+]i) and muscarinic receptors (to generate DAG). This demonstrates an unusual form of synergism between the two effects of a single transmitter mediated via separate receptors operating within a time scale that could be of physiological significance.
Asunto(s)
Calcio/metabolismo , Canales de Cloruro/fisiología , Neuronas/fisiología , Proteína Quinasa C/metabolismo , Receptores Muscarínicos/fisiología , Acetilcolina/farmacología , Animales , Calcio/farmacología , Diglicéridos/farmacología , Sinergismo Farmacológico , Conductividad Eléctrica , Ganglios Simpáticos/citología , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Receptores Muscarínicos/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/fisiologíaRESUMEN
The properties of neuronal-type nicotinic acetylcholine receptors (nAChRs) present on the neuroendocrine cells of the porcine pars intermedia of the pituitary were studied in intact single cell using measurements of the free intracellular Ca2+ concentration ([Ca]i) with the calcium-sensitive dye fura 2. Local application of an extracellular solution containing 50 mM K+ or of the selective nAChR agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP) depolarised the cells and induced an elevation in [Ca]i. The effect of DMPP on [Ca]i was dose dependent (EC50 = 6 microM), reversibly blocked by d-tubocurarine and strictly dependent on the concentration of extracellular Ca2+. The calcium channel blocker Cd2+ (100 microM) reversibly blocked 80% of the response induced by 50 mM K+, whereas it reduced the DMPP response by only 50%. In the absence of extracellular Na+, DMPP no longer depolarised the cells but still increased [Ca]i. The rise in [Ca]i under these conditions represented 41% of the control response, i.e. in the presence of external Na+. Thus activation of nAChRs induced an elevation in [Ca]i which was in part independent of cell depolarisation. This was confirmed by recording simultaneously, under whole-cell voltage-clamp, a rise in [Ca]i associated with the inward nicotinic current. During prolonged application of the agonist (50 s), the amplitude of the nicotinic current decayed rapidly to a very low plateau level reflecting nAChR desensitisation. However, photometric experiments performed on intact non-dialysed cells revealed the presence of a slowly decaying phase in [Ca]i throughout the application of DMPP. This suggests the persistence of a substantial Ca2+ influx during prolonged exposure to the agonist. Taken together, our results show that stimulation of nAChRs induces an influx of Ca2+ which elevates [Ca]i. This phenomenon is due to activation of voltage-dependent Ca2+ channels and to Ca2+ entry through the nAChR.
Asunto(s)
Calcio/metabolismo , Neuronas/metabolismo , Hipófisis/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Canales de Calcio/metabolismo , Células Cultivadas , Yoduro de Dimetilfenilpiperazina/farmacología , Electrofisiología , Femenino , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Fotometría , Hipófisis/citología , Hipófisis/efectos de los fármacos , Potasio/farmacología , Receptores Nicotínicos/efectos de los fármacos , PorcinosRESUMEN
Variations in intracellular free calcium concentration (delta[Ca2+]i) were measured in intact and isolated human astrocytoma cells (U373 MG) loaded with fura-2 acetoxymethylester. Microperfusion of 50 nM substance P (SP), applied for 1 s, increased [Ca2+]i by 351 nM from a stable basal level of [Ca2+]i of 26 nM. The peak delta[Ca2+]i induced by SP was dose dependent with a threshold of 10(-3) nM, an ED50 of 1.3 nM and a maximal effect for concentrations of SP greater than 100 nM. The NK1 receptor agonist, [Sar9Met(O2)11]SP, mimicked the effect of SP, while the NK2 and NK3 selective receptor agonists, [N1(10)]NKA(4-10) and senktide, respectively, had no effect. The delta[Ca2+]i induced by SP was unaffected by 100 microM cadmium or by removal of extracellular calcium ions. Caffeine up to 30 mM had no effect on [Ca2+]i. In contrast, thapsigargin increased resting [Ca2+]i by 92 nM and reduced the delta[Ca2+]i induced by SP. A pertussis treatment (500 ng/ml-24 h) did not modify the delta[Ca2+]i induced by SP. We conclude that SP, acting on a NK1 receptor, mobilizes cytosolic calcium from an intracellular calcium pool which can be partially depleted by thapsigargin.
Asunto(s)
Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Calcio/metabolismo , Sustancia P/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Fura-2 , Humanos , Antagonistas del Receptor de Neuroquinina-1 , Receptores de Neuroquinina-1/agonistas , Receptores de Neuroquinina-2/agonistas , Receptores de Neuroquinina-2/antagonistas & inhibidores , Receptores de Neuroquinina-3/agonistas , Receptores de Neuroquinina-3/antagonistas & inhibidores , Células Tumorales Cultivadas , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Intracellular free calcium concentration ([Ca2+]i) was measured in cultured explants of myenteric plexus neurones by using the fluorescent calcium indicator Indol in combination with patch-clamp techniques. The basal [Ca2+]i was 94 nM and spontaneous oscillations in the internal free calcium concentration were recorded. These oscillations were associated with bursts of action potentials triggered by spontaneous nicotinic excitatory synaptic potentials. Under voltage clamp conditions, application of the selective nicotinic agonist m-hydroxyphenylpropyl-trimethylammonium iodide (10 microM) induced an inward current and increased the intracellular free calcium concentration. We conclude that cholinergic synaptic excitatory activity provide a regular calcium entry in myenteric neurone and suggest that the nicotinic channel might be significantly permeable to calcium.
Asunto(s)
Calcio/metabolismo , Membranas Intracelulares/metabolismo , Plexo Mientérico/metabolismo , Neuronas/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Células Cultivadas , Estimulación Eléctrica , Electrofisiología , Potenciales Evocados , Plexo Mientérico/citología , Plexo Mientérico/fisiología , Neuronas/fisiología , Concentración Osmolar , Estimulación QuímicaRESUMEN
1. Patch-clamp techniques in conjunction with indo-1 fluorescent measurements were used to measure increases in intracellular free calcium concentration and membrane conductance induced by the activation of nicotinic and calcium channels in cultured rat sympathetic neurons. 2. Under voltage-clamp conditions, pressure application of the nicotinic agonist DMPP (1,1-dimethyl-4-phenylpiperazinium iodide, 100 microM, 100 ms) increased [Ca2+]i by 193 +/- 26 nM at a clamp potential of -60 mV. This was accompanied by an inward current of -4.53 +/- 0.89 nA, giving a mean ratio of the delta (Ca2+]i to the total inward charge transfer of 42.7 nmoles per litre of free calcium per nanocoulomb of charge (M/q ratio). 3. The DMPP-induced current and associated delta [Ca2+]i were reduced by mecamylamine (100 nM-10 microM) but were unaffected by alpha-bungarotoxin (100 nM) or cadmium (100 microM). 4. The M/q ratio was not affected by the holding potential (from -80 to -40 mV) but was a function of the external calcium concentration. 5. The M/q ratio was reduced by increasing the intracellular calcium buffering capacity and increased by heparin but not affected by ryanodine or by depletion of the caffeine-sensitive calcium store. 6. Under the same recording conditions, we quantified the increase in [Ca2+]i associated with activation of the voltage-dependent calcium current. On average at -60 mV, the M/q ratio of this highly calcium-selective permeability was 1961 mM nC-1, which is 46 times that obtained for the nicotinic channel. 7. Assuming constant-field theory, ion-substitution experiments suggest that in 2.5 mM external calcium, the permeability sequence for the nicotinic conductance was Cs+ < Li+ < Na+ < K+ < Ca2+. 8. We conclude that the nicotinic channels in rat sympathetic neurones are significantly permeant to Ca2+ and that the influx of Ca2+ through these channels is the principal cause of the rise in [Ca2+]i seen under voltage clamp.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Receptores Nicotínicos/metabolismo , Ganglio Cervical Superior/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Canales de Calcio/efectos de los fármacos , Células Cultivadas , Yoduro de Dimetilfenilpiperazina/farmacología , Homeostasis , Líquido Intracelular/metabolismo , Potenciales de la Membrana , Permeabilidad , Ratas , Receptores Nicotínicos/efectos de los fármacos , Ganglio Cervical Superior/citología , Ganglio Cervical Superior/efectos de los fármacosRESUMEN
1. The M-like current IK(M,ng) in differentiated NG108-15 mouse neuroblastoma x rat glioma hybrid cells has been studied using tight-seal, whole-cell patch-clamp recording. 2. When calculated from steady-state current-voltage curves, the conductance underlying IK(M,ng) showed a Boltzmann dependence on voltage with half-activation voltage Vo = -44 mV (in 3 mM [K+]) and slope factor (a) = 8.1 mV/e-fold increase in conductance. In 12 mM [K+] Vo = -38 mV and a = 6.9 mV. The deactivation reciprocal time constant accelerated with hyperpolarization with slope factor 17 mV/e-fold voltage change. 3. The reversal potential for deactivation tail currents varied with external [K+] as if PNa/PK were 0.005. 4. Steady-state current was increased on removing external Ca2+. In the presence of external Ca2+, reactivation of IK(M, ng) after a hyperpolarizing step was delayed. This delay was preceded by an inward Ca2+ current, and coincided with an increase in intracellular [Ca2+] as measured with Indo-1 fluorescence. Elevation of intracellular [Ca2+] with caffeine also reduced IK(M, ng). 5. IK(M, ng) was inhibited by external divalent cations in decreasing order of potency (mM IC50 in parentheses): Zn2+ (0.011) greater than Cu2+ (0.018) greater than Cd2+ (0.070) greater than Ni2+ (0.44) greater than Ba2+ (0.47) greater than Fe2+ (0.69) greater than Mn2+ (0.86) greater than Co2+ (0.92) greater than Ca2+ (5.6) greater than Mg2+ (16) greater than Sr2+ (33). This was not secondary to inhibition of ICa since: (i) inhibition persisted in Ca(2+)-free solution; (ii) La3+ did not inhibit IK(M, ng) at concentrations which inhibited ICa; and (iii) organic Ca2+ channel blockers were ineffective. Inhibition comprised both depression of the maximum conductance and a positive shift of the activation curve. Addition of Ca2+ (10 microM free [Ca2+]) or Ba2+ (1 mM total [Ba2+]) to the pipette solution did not significantly change IK(M, ng). 6. IK(M, ng) was reduced by 9-amino-1,2,3,4-tetrahydroacridine (IC50 8 microM) and quinine (30 microM) but was insensitive to tetraethylammonium (IC50 greater than 30 mM), 4-aminopyridine (greater than 10 mM), apamin (greater than 3 microM) or dendrotoxin (greater than 100 nM). 7. IK(M, ng) was inhibited by bradykinin (1-10 microM) or angiotensin II (1-10 microM), but not by the following other receptor agonists: acetylcholine (10 mM), muscarine (10 microM), noradrenaline (100 microM), adrenaline (100 microM), dopamine (100 microM), histamine (100 microM), 5-hydroxytryptamine (10 microM), Met-enkephalin (1 microM), glycine (100 microM), gamma-aminobutyric acid (100 microM) or baclofen (500 microM).(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Neuronas/metabolismo , Potasio/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Calcio/metabolismo , Calcio/farmacología , Cationes Bivalentes/farmacología , Glioma/metabolismo , Células Híbridas/efectos de los fármacos , Células Híbridas/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Ratones , Neuroblastoma/metabolismo , Neuronas/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , RatasRESUMEN
The electrophysiological properties of porcine melanotrophs in primary culture were studied with patch clamp techniques. In the cell-attached (c/a) configuration, extracellular spikes were recorded but in only 47 of the 259 cells examined; active cells were more frequently found in short-term cultures (48 h, 22 out of 71). In the whole-cell (WCR) configuration, the mean resting potential was -46.6 mV and the mean input resistance 2.41 G omega. In the current clamp mode, of the 99 cells recorded, 39.4% displayed spontaneous spiking activities, 14.1% spiked when a depolarizing current was applied, and 46.5% were silent. Two patterns of spontaneous activity were recorded. The first consisted of rapid membrane depolarizations (mean duration: 16.0 ms) which reached a mean value of +8.9 mV. These action potentials occurred either at random intervals or at a mean frequency of 1.19 Hz. Tetrodotoxin (TTX), a sodium channel blocker, completely abolished these action potentials. The second pattern consisted of regular bursts (mean duration: 1.69 s and mean frequency: 0.15 Hz) of spikes with smaller amplitudes (culminating at -9.5 mV) and greater durations (79.7 ms). This pattern could be recorded in the presence of TTX in the external medium. In the c/a configuration, thyrotropin-releasing hormone (TRH) and (3Me-His2)-TRH enhanced spike frequency, whereas histidyl-proline-diketopiperazine, a metabolite of TRH, had no effect. In WCR, out of the 27 cells tested, TRH (5.10(-9)-5.10(-8) M) induced firing in 4 quiescent cells and increased the frequency of action potentials in 4 spontaneously active cells. This was usually, but not necessarily, preceded by a hyperpolarization (n = 5). TRH (5.10(-8) M) enhanced the secretion of melanocyte-stimulating hormone (alpha MSH) from perfused isolated melanotrophs by 62.8 +/- 9.8% (n = 9) over basal levels. (3Me-His2)-TRH and (Pro3)-TRH mimicked the TRH response, whereas histidyl-proline-diketopiperazine was without effect.
Asunto(s)
Hipotálamo Medio/fisiología , Hormona Liberadora de Tirotropina/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Células Cultivadas , Femenino , Hipotálamo Medio/efectos de los fármacos , Hipotálamo Medio/metabolismo , Inmunohistoquímica , Técnicas In Vitro , Masculino , Hormonas Estimuladoras de los Melanocitos/sangre , Hormonas Estimuladoras de los Melanocitos/inmunología , Porcinos , Tetrodotoxina/farmacologíaRESUMEN
The action of thyrotropin-releasing hormone (TRH) on melanotrope cells maintained in primary culture was studied with biochemical and electrophysiological techniques. TRH effects on polyphosphoinositide (PPI) breakdown was measured in [3H]myoinositol labelled cells maintained in suspension for 24 hours or in primary culture. TRH (50 nM) or its potent analogue (3Me-His2)-TRH increased total PPI levels by 50-125% in separate experiments after 30 min of treatment whereas corticotropin-releasing hormone (CRF) was without effect. The effect of TRH was dose-dependent (ED50 = 5 nM), the maximal effect being reached with 50 nM TRH. Using the patch-clamp technique in the cell-attached configuration spikes were recorded extracellularly. In 6 of the 13 cells tested, (3Me-His2)-TRH (10 nM) elicited an increase in the spontaneous spiking rate. Furthermore, TRH (50 nM) increased melanocyte-stimulating hormone (alpha-MSH) secretion 2-fold after 8 h of treatment. These results suggested that TRH activated phospholipase C and electrical activity in melanotrope cells; the resulting phosphoinositide breakdown and increase in intracellular free Ca2+ ultimately led to a stimulation of hormone release.
Asunto(s)
Fosfatos de Inositol/metabolismo , Melanocitos/fisiología , Receptores de Neurotransmisores/fisiología , Fosfatos de Azúcar/metabolismo , Hormona Liberadora de Tirotropina/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Células Cultivadas , Técnicas In Vitro , Melanocitos/citología , Melanocitos/efectos de los fármacos , Receptores de Neurotransmisores/efectos de los fármacos , Receptores de Hormona Liberadora de Tirotropina , PorcinosRESUMEN
On porcine intermediate lobe (IL) endocrine cells, spontaneously opening chloride channels have been studied and compared to GABA-A activated chloride channels. Elementary currents were recorded mainly from outside-out patches excised from IL cells maintained in culture for 1-4 weeks. Spontaneous inward currents were observed in Cs-loaded cells after replacing Na in the extracellular medium by the impermeant ion choline. This activity, at an internal calcium concentration of 10(-8) M corresponded to a channel for chloride ions with a main conductance level of 26 pS, and substates around 11 pS. The sequence of permeabilities to halides was I greater than Br greater than Cl. These conductance characteristics were common to the GABA-operated channels which also showed a main conductance substate of 23-31 pS. The open time of the 26 pS level mostly encountered in spontaneous activity, was distributed along two modes: one, the most frequent, around 1 ms, and the other around 4 ms. This latter mode was the predominant one observed during GABA and isoguvacine applications but in addition a bursting activity of 19 ms duration was also seen. Specific GABA-A receptor antagonists (bicuculline and SR42641, 1 microM) blocked activity evoked by GABA (1-10 microM), but did not affect spontaneous events. These spontaneous Cl events were only observed in a restricted range of internal Ca concentrations, i.e. between 1 nM and 0.1 microM, and were practically abolished at Cai 1 microM. The GABA-induced activity of Cl channels was also Ca-sensitive, being reduced when Cai reached 1 microM.
Asunto(s)
Canales Iónicos/fisiología , Hipófisis/metabolismo , Ácido gamma-Aminobutírico/farmacología , Animales , Calcio/fisiología , Células Cultivadas , Cloruros/metabolismo , Canales Iónicos/efectos de los fármacos , Iones/metabolismo , Ácidos Isonicotínicos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Hipófisis/efectos de los fármacos , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/fisiologíaRESUMEN
The inhibitory neurotransmitter GABA activates two receptor subtypes that can be distinguished by their pharmacology. The GABA-A site is competitively antagonized by bicuculline and exclusively coupled to a chloride channel. The GABA-B receptor, for which baclofen is the only specific agonist, is resistant to bicuculline inhibition and, depending upon its localization, will activate K currents and/or inhibit Ca currents. Both electrophysiological and biochemical approaches have been applied to the study of each receptor. The membrane and intracellular components that to date have been implicated in GABA-B activation are discussed: G proteins, adenylate cyclase and intracellular calcium levels. This latter factor is also discussed with respect to GABA-A receptor action.
Asunto(s)
Receptores de GABA-A/efectos de los fármacos , Calcio/fisiología , Canales Iónicos/efectos de los fármacos , Proteínas del Tejido Nervioso/fisiologíaRESUMEN
We have examined the action of GABA on the electrical, secretory and synthetic activities of rat and porcine intermediate lobe (IL) cells in primary culture. Chloride and calcium currents were investigated using patch-clamp techniques. A chloride current activated by 1-100 microM isoguvacine, a specific GABA-A agonist, and antagonised by bicuculline and SR 95103 was recorded at the whole cell and single channel level current. Whole cell calcium currents were investigated and shown to be reduced by 40 microM cadmium, zero external calcium and 10 microM baclofen, a specific GABA-B receptor agonist. Both GABA-B receptor activation and use of calcium deficient medium inhibited peptide release from IL cells. Finally, pro-opiomelanocortin (POMC) mRNA levels were measured using a hybridization technique. Removal of calcium from the culture medium or long-term (48 hr) incubation with 10 microM GABA or muscimol (a mixed GABA-A and GABA-B agonist) significantly reduced POMC mRNA levels.