RESUMO
Ptychography is a scanning coherent diffractive imaging (CDI) technique that relies upon a high level of stability of the illumination during the course of an experiment. This is particularly an issue for coherent short wavelength sources, where the beam intensity is usually tightly focused on the sample in order to maximize the photon flux density on the illuminated region of the sample and thus a small change in the beam position results in a significant change in illumination of the sample. We present an improved ptychographic method that allows for limited stability of the illumination wavefront and thus significantly improve the reconstruction quality without additional prior knowledge. We have tested our reconstruction method in a proof of concept experiment, where the beam instability of a visible light source was emulated using a piezo driven mirror, and also in a short wavelength microscopy CDI setup using a high harmonic generation source in the extreme ultraviolet range. Our work shows a natural extension of the ptychography method that paves the way to use ptychographic imaging with any limited pointing stability coherent source such as free electron or soft X-ray lasers and improve reconstruction quality of long duration synchrotron experiments.
RESUMO
Repetitive sensory input, stroboscopic lights or repeated sounds can induce epileptic seizures in susceptible individuals. In order to understand the process we have to consider multiple factors. The output of a set of neurones is determined by the amount of excitatory synaptic input, the degree of positive feedback and their inherent electrical excitability, which can be modified by synaptic inhibition. Recent research has shown that it is possible to separate these phenomena, and that they do not always behave in unison.
Assuntos
Epilepsia/fisiopatologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios Aferentes/fisiologia , Sinapses/fisiologia , Animais , Vias Auditivas/citologia , Humanos , Vias Visuais/citologiaRESUMO
Standard two electrode voltage clamp techniques were used to investigate the response of neuronal nicotinic acetylcholine receptors, expressed in Xenopus oocytes, to various agonists and neuronal bungarotoxin (NBT). The beta subunit is an important determinant of the receptor's pharmacological profile. Co-expression of alpha 4 and beta 2 subunits produced a receptor that was relatively insensitive to cytisine and nicotine and inhibited by NBT, whilst the alpha 4 beta 4 combination produced a receptor that was highly sensitive to cytisine and nicotine but resistant to toxin. The first 80 amino acids of the N-terminal domain of the beta subunit are implicated in these characteristics, since the combination of alpha 4 with a hybrid beta subunit comprising amino acids 1-->80 of beta 2 and 81-->416 of beta 4 became relatively insensitive to nicotine and cytisine and resistant to inhibition by neuronal bungarotoxin.
Assuntos
Bungarotoxinas/farmacologia , Neurônios/química , Receptores Nicotínicos/efeitos dos fármacos , Acetilcolina/farmacologia , Alcaloides/farmacologia , Animais , Azocinas , Sítios de Ligação , Reagentes de Ligações Cruzadas , Iodeto de Dimetilfenilpiperazina/farmacologia , Glicosilação , Nicotina/farmacologia , Quinolizinas , Receptores Nicotínicos/química , XenopusRESUMO
The signalling pathways by which insulin triggers protein synthesis were studied using an antisense strategy to deplete ERK1/ERK2 and rapamycin to inhibit the p70S6K pathway. The results indicated that ERK1/ERK2 principally regulated the amount of the protein synthesis machinery available in the cell while the p70S6K pathway contributed to modulating its activation in response to insulin. ERK1/ERK2 also mediated in a small proportion of insulin-stimulated protein synthesis which included the induction of c-fos protein. When c-fos induction was blocked the majority of insulin-stimulated protein synthesis still occurred and thus did not require transcriptional regulation of c-fos or its targets.
Assuntos
Adipócitos/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Proteínas Quinases Ativadas por Mitógeno , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Hipoglicemiantes/metabolismo , Insulina/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Biossíntese de ProteínasRESUMO
Unilateral kainic acid lesion in the hippocampus caused a long-term change in the balance between excitatory and inhibitory drive onto CA1 pyramidal cells, making these cells hyperexcitable several weeks post-lesion. In this study, we have shown an enhanced N-methyl-D-aspartate receptor-mediated component in the excitatory synaptic transmission together with a reduced GABA(A) receptor-mediated inhibition in CA1 pyramidal cells one-week post kainic acid lesion. In these cells, pharmacologically isolated N-methyl-D-aspartate receptor-mediated whole-cell excitatory postsynaptic currents were significantly larger at negative holding potentials, and the voltage-dependence of N-methyl-D-aspartate receptor channels was shifted in the hyperpolarizing direction. The plot of relative conductance (g/gMax) shifted significantly (P<0.01) to more negative holding potentials by 19 mV (-28+/-4 mV in control slices and -47+/-4 mV in kainic acid slices) at the half maximal conductance point (g/gMax =0.5). This shift gives a larger N-methyl-D-aspartate receptor-mediated component in the excitatory synaptic transmission at resting membrane potentials (around -60 mV). The shifted voltage dependence is highly sensitive to extracellular Mg2+ ions. Moderate increases in [Mg2+]o from 1 mM to 2.6 mM more than compensated for the negative shift and effectively suppressed the population epileptiform bursting activity. Fitting the voltage dependence to an ionic block model revealed a higher dissociation constant of N-methyl-D-aspartate receptor channels for Mg2+ in kainic acid-lesioned slices (52 mM at 0 mV; 330 microM at -60 mV) than in control slices (7.7 mM at 0 mV; 93 microM at -60 mV). While a simple single site model adequately fitted the control data for [Mg2+]o at 1 mM and 2.6 mM, no consistent model of this form was found for the kainic acid-lesioned slices. These results revealed changed properties of N-methyl-D-aspartate receptor channels in the kainic acid-lesioned model of epilepsy. The reduced Mg2+ blockade of N-methyl-D-aspartate receptor channels contributed significantly to the epileptiform bursting activity.
Assuntos
Potenciais Evocados/fisiologia , Hipocampo/fisiologia , Ácido Caínico/farmacologia , Magnésio/farmacologia , Células Piramidais/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Transmissão Sináptica/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Estimulação Elétrica , Potenciais Evocados/efeitos dos fármacos , Hipocampo/citologia , Técnicas In Vitro , Lidocaína/análogos & derivados , Lidocaína/farmacologia , Masculino , Modelos Neurológicos , Técnicas de Patch-Clamp , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Ratos , Ratos Wistar , Valores de Referência , Transmissão Sináptica/efeitos dos fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
1. The action of 5-hydroxytryptamine (5-HT) on neuronal and muscle nicotinic acetylcholine receptors (nicotinic AChR) expressed in Xenopus oocytes was studied. 2. 5-HT enhanced the rate of desensitization of the acetylcholine (ACh) current response in all receptor subtypes investigated (muscle, alpha beta 2 gamma delta and alpha 4 beta 2), acting in a dose-dependent manner. 3. 5-HT also reduced the peak current elicited by ACh in a dose-dependent manner. The IC50 value for the muscle type receptor was 227 +/- 0.44 microM, and 166 +/- 0.47 microM and 283 +/- 0.28 microM for the combinations alpha beta 2 gamma delta and alpha 4 beta 2 respectively. 4. The effect of 5-HT on the responses to ACh (10 microM) was found to be independent of membrane voltage over the range tested (-80 to -10 mV), and to be readily reversed by washout. 5. The action of 5-HT could be mimicked by structurally similar molecules. The homologue tryptamine was less potent than 5-HT in blocking the ACh current, with an IC50 of 1.0 +/- 0.02 mM. Ketanserin, a 5-HT2 receptor antagonist, was more potent than 5-HT, the IC50 being 49.0 +/- 1.4 microM. 6. We postulate that a highly conserved portion of the tertiary structure of nicotinic AChRs, which includes some part of the ACh binding site, has affinity for 5-HT and structural analogues.
Assuntos
Potenciais da Membrana/efeitos dos fármacos , Músculos/metabolismo , Neurônios/metabolismo , Receptores Nicotínicos/biossíntese , Serotonina/farmacologia , Acetilcolina/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Músculos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oócitos , RNA Mensageiro/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Triptaminas/farmacologia , Xenopus laevisRESUMO
1. The aim of these experiments was to determine the ability of the muscle-type nicotinic acetylcholine receptor (AChR) stably expressed in quail fibroblasts (QF18 cells) to elevate intracellular calcium ([Ca2+]i) upon activation. Ratiometric confocal microscopy, with the calcium-sensitive fluorescent dye Indo-1 was used. 2. Application of the nicotine agonist, suberyldicholine (SDC), to the transfected QF18 cells caused an increase in [Ca2+]i. Control [Ca2+]i levels in QF18 cells were found to be 164 +/- 22 nM (mean +/- s.e. mean; n = 40 cells) rising to 600 +/- 81 nM on addition of SDC (10 microM; n = 15 cells), whereas no increase in [Ca2+]i was seen in non-transfected control QT6 fibroblasts (before: 128 +/- 9 nM, n = 40; after; 113 +/- 13 nM, n = 15). 3. The increase in [Ca2+]i caused by application of SDC was dose-dependent, with an EC50 value of 12.7 +/- 5.9 microM (n = 14). 4. The responses to SDC in QF18 cells were blocked by prior application of alpha-bungarotoxin (200 nM), by the addition of Ca2+ (100 microM), by removal of Na+ ions from the extracellular solution, or by the voltage-sensitive calcium channel blockers nifedipine and omega-conotoxin, which act with IC50 values of 100 nM and 100 pM respectively. 5. We conclude that activation of the nicotinic AChRs leads to a Na(+)-dependent depolarization and hence activation of endogenous voltage-sensitive Ca2+ channels in the plasma membrane and an increase in [Ca2+]i. There is no significant entry of Ca2+ through the nicotinic receptor itself.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Agonistas Nicotínicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Colina/análogos & derivados , Colina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Microscopia Confocal , Nifedipino/farmacologia , Peptídeos/farmacologia , Codorniz , Reprodutibilidade dos Testes , Transfecção , ômega-Conotoxina GVIARESUMO
The effects of several modulators of ryanodine receptors (RYRs) on the reduction of acetylcholine induced inward current (ACh-current) evoked by EPYLRFamide (5 microM, bath application), the potent N-terminally modified analogue of the endogenous Helix heptapeptide SEPYLRFamide, were investigated. These modulators were applied intracellularly. Inward currents were recorded from identified Helix lucorum LPa2, LPa3, RPa3, RPa2 neurones in ganglia preparations using the two-electrode voltage clamp technique. ACh was applied ionophoretically. BAPTA (0.1 mM), chelator of intracellular Ca(2+), ryanodine (0.1 mM), agonist/antagonist of RYRs and dantrolene (0.1 mM), antagonist of RYRs decrease the effect of EPYLRFamide. Adenosine (1 mM), alpha,beta-methylene ATP (0.1 mM), the nonhydrolisable ATP analogue and cyclic adenosine diphosphate ribose (0.1 mM) (agonists of RYRs) potentiate the modulatory effect of EPYLRFamide. Ruthenium red (1 mM), antagonist of RYRs and caffeine (1 mM), agonist of RYRs do not change the modulatory effect of EPYLRFamide. These data suggest that intracellular Ca(2+) and RYRs are involved in the modulatory effect of EPYLRFamide on ACh-currents. It was concluded that EPYLRFamide decreases ACh-current through elevation of basal intracellular level of a putative endogenous agonist of RYRs which activates RYR-dependent mobilization of Ca(2+) by binding to the adenine nucleotide site of the ryanodine receptor-channel complex and does not bind the site activated by caffeine.
Assuntos
Acetilcolina/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Adenosina/farmacologia , Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Compostos de Anilina , Animais , Cafeína/farmacologia , Quelantes/farmacologia , ADP-Ribose Cíclica , Dantroleno/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Corantes Fluorescentes , Caracois Helix , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Rianodina/farmacologia , XantenosRESUMO
We have investigated the relative contributions of oxygen and glucose deprivation to ischaemic neurodegeneration in organotypic hippocampal slice cultures. Cultures prepared from 10-day-old rats were maintained in vitro for 14 days and then deprived of either oxygen (hypoxia), glucose (hypoglycaemia), or both oxygen and glucose (ischaemia). Hypoxia alone induced degeneration selectively in CA1 pyramidal cells and this was greatly potentiated if glucose was removed from the medium. We have also characterised the effects of both pre- and post-treatment using glutamate receptor antagonists and the sodium channel blocker tetrodotoxin (TTX). Neuronal death following either hypoxia or ischaemia was prevented by pre-incubation with CNQX, MK-801 or tetrodotoxin. MK-801 or CNQX also prevented death induced by either hypoxia or ischaemia if added immediately post-insult, however, post-insult addition of TTX prevented hypoxic but not ischaemic damage. Organotypic hippocampal slice cultures are sensitive to both NMDA and non-NMDA glutamate receptor blockade and thus represent a useful in vitro system for the study of ischaemic neurodegeneration paralleling results reported using in vivo models of ischaemia.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Isquemia Encefálica/patologia , Morte Celular/efeitos dos fármacos , Hipocampo/irrigação sanguínea , Hipoglicemia/tratamento farmacológico , Hipoglicemia/patologia , Hipóxia Encefálica/tratamento farmacológico , Hipóxia Encefálica/patologia , Neurônios/patologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Receptores de AMPA/antagonistas & inibidores , Canais de Sódio/efeitos dos fármacosRESUMO
The inhibition of CA1 pyramidal neurones in rat hippocampal slices was studied using extracellular recordings of population spike potential responses to paired orthodromic stimulation. Variation of the interpulse interval allowed the separation of an early phase of inhibition (interpulse interval 5-20 ms), blocked by the GABAA receptor antagonist bicuculline (1 microM; n = 11), and a late phase (interpulse interval 200-400 ms) blocked by the GABAB receptor antagonist phaclofen (1 mM; n = 5) but enhanced by bicuculline (n = 11). Similar enhancement was not observed when conditioning response amplitudes were increased by increasing the stimulus strength, rather than bicuculline. Orthodromic stimulation leads to synaptic excitation of both pyramidal neurones and inhibitory interneurones, and may also lead to activation of inhibitory inputs onto interneurones. Bicuculline could prevent inhibition of the interneurones, and hence enhance the late, GABAB receptor-mediated inhibition. Conversely, the therapeutic administration of benzodiazepines would be postulated to enhance the inhibition of inhibitory interneurones, leading to an iatrogenic decrease in GABAB receptor-mediated inhibition.
Assuntos
Baclofeno/análogos & derivados , Bicuculina/farmacologia , Antagonistas GABAérgicos/farmacologia , Hipocampo/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Receptores de GABA-B/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Baclofeno/farmacologia , Estimulação Elétrica , Hipocampo/citologia , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de GABA-B/metabolismoRESUMO
2-Hydroxy-saclofen is known to be active at GABAB receptors in the mammalian central nervous system, and we have investigated its effects on synaptic transmission in the rat hippocampal slice preparation. Orthodromic stimuli were applied to the stratum radiatum, and population spike responses from the CA1 pyramidal cell layer were recorded extracellularly. A second, identical stimulus was applied at a variable interpulse interval (IPI) after the initial conditioning stimulus. GABAergic synaptic inhibition was observed as a decrease in the spike amplitude of the second response compared to the first. Both the GABAB receptor antagonist phaclofen (1 mM) and 2-hydroxy-saclofen (200 microM) prevented a slow phase of inhibition for IPIs of 200-400 ms. However, these agents differed markedly in their effects on overall synaptic transmission. Phaclofen had no effect on the amplitude of the initial conditioning spike amplitude, whereas 2-hydroxy-saclofen reduced it significantly, in a manner similar to baclofen (1 microM). The direct actions of 2-hydroxy-saclofen were unexpected for a pure antagonist of GABAB receptors, but could be prevented by the co-administration of phaclofen (1 mM), but not bicuculline (1 microM). Reduction in conditioning spike amplitude due to antagonism of GABAB autoreceptors on inhibitory interneurones and subsequent enhancement of GABAA tonic inhibition would have been blocked by bicuculline. The blockade of the 2-hydroxy-saclofen effect by phaclofen implies a GABAB receptor partial agonist action. The possible sites of this action are discussed.
Assuntos
Baclofeno/análogos & derivados , Antagonistas GABAérgicos/farmacologia , Hipocampo/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Baclofeno/farmacologia , Bicuculina/farmacologia , Potenciais Evocados/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Microeletrodos , Técnicas de Patch-Clamp , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de GABA-B/efeitos dos fármacosRESUMO
The pharmacological diversity of the different isoforms of the nicotinic acetylcholine receptor arises from the diversity of the subunits that assemble to form the native receptors. The aim of this study was to investigate the actions of the muscle relaxants d-tubocurarine, pancuronium and vecuronium on different isoforms of nicotinic acetylcholine receptors (mouse foetal muscle, mouse adult muscle and a rat neuronal), using the Xenopus oocyte expression system. Oocytes were injected with cRNAs for alpha, beta, gamma, delta subunits (the native foetal muscle subunit combination), or with cRNAs for alpha, beta, epsilon, delta subunits (the native adult muscle subunit combination), or with cRNAs for alpha4beta2 subunits (a putative native neuronal subunit combination). Acetylcholine had a similar potency at all three subunit combinations (EC50 11.6, 17.4 and 19.1 microM, respectively). At all three receptor types, d-tubocurarine and pancuronium blocked the responses elicited by acetylcholine in a reversible manner. Furthermore, the inhibition of the acetylcholine currents for the foetal and adult nicotinic acetylcholine receptor by pancuronium and d-tubocurarine was independent of the holding voltage over the range -100 to -40 mV. In oocytes expressing the foetal muscle nicotinic acetylcholine receptors the inhibition of the current in response to 100 microM acetylcholine by 10 nM d-tubocurarine was 29 +/- 5% (mean +/- S.E.M.; n = 7), and the inhibition by 10 nM pancuronium was 39 +/- 6% (mean +/- S.E.M.; n = 8; P > 0.05 vs. d-tubocurarine). However, in the adult form of the muscle nicotinic acetylcholine receptor, 10 nM d-tubocurarine and 10 nM pancuronium were both more effective at blocking the response to 100 microM acetylcholine compared to the foetal muscle nicotinic acetylcholine receptor, with values of 55 +/- 5% (P < 0.01; n = 12) and 60 +/- 4% (P < 0.001; n = 10), respectively. Thus the developmental switch from the gamma to the epsilon subunit alters the antagonism of the nicotinic acetylcholine receptor for both pancuronium and d-tubocurarine. Vecuronium was more potent than pancuronium. One nM vecuronium reduced the response to 100 microM acetylcholine by 71 +- 6% (n = 10) for foetal and 63 +/- 5% (n = 4) for adult nicotinic acetylcholine receptors. In the alpha4beta2 neuronal nicotinic acetylcholine receptor combination, 10 nM pancuronium was a more effective antagonist of the response to 100 microM acetylcholine (69 +/- 6%, n = 6) than 10 nM d-tubocurarine (30 +/- 5%; n = 6; P < 0.05 compared to pancuronium). This is in contrast to the adult muscle nicotinic acetylcholine receptor, where pancuronium and d-tubocurarine were equieffective. The expression of the beta2 subunit with muscle alpha, epsilon and delta subunits formed a functional receptor which was blocked by pancuronium and d-tubocurarine in a similar manner to the alphabeta1epsilondelta subunit consistent with the hypothesis that the beta subunit is not a major determinant in the action of this drug at the adult muscle nicotinic acetylcholine receptor.
Assuntos
Fármacos Neuromusculares não Despolarizantes/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Feminino , Camundongos , Músculos/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Pancurônio/farmacologia , Técnicas de Patch-Clamp , Isoformas de Proteínas , Ratos , Receptores Nicotínicos/química , Receptores Nicotínicos/classificação , Tubocurarina/farmacologia , Brometo de Vecurônio/farmacologia , Xenopus laevisRESUMO
In this paper we report our observations of the relationship of paired-pulse facilitation (PPF) with synaptic release or release failure at small numbers of synaptic sites. Minimal stimulation protocols were employed to enable the activation of only one or a few axons which synapse onto CA1 pyramidal cells in the hippocampus. Excitatory postsynaptic currents (EPSCs) in response to paired stimulation were measured. On the analysis of the data, we examined the effects of failure and synaptic release on PPF, and found that PPF was observed as a decrease in failures of synaptic release in response to test stimuli which were preceded by conditioning stimuli that evoked synaptic release. The test-pulse failure rate following conditioning pulse failures was indistinguishable from the overall conditioning pulse failure rate. It is postulated that the mechanism of paired-pulse facilitation is presynaptic and associated with successful synaptic release in response to the conditioning stimulus.
Assuntos
Hipocampo/citologia , Neurônios/fisiologia , Transmissão Sináptica/fisiologia , Animais , Estimulação Elétrica , Hipocampo/fisiologia , Masculino , Plasticidade Neuronal/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos WistarRESUMO
The acute toxicity of organophosphorus (OP) nerve agents arises from accumulation of acetylcholine (ACh) and overstimulation of ACh receptors. The mainstay of current pharmacotherapy is the competitive muscarinic antagonist, atropine. Nicotinic antagonists have not been used due to the difficulties of administering a dose of a competitive neuromuscular blocker sufficient to antagonise the effects of excessive ACh, but not so much that it paralyses the muscles. An alternative approach would be to use a noncompetitive antagonist whose effects would not be overcome by increasing ACh concentrations. This study demonstrates that the compound 1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium), which blocks open nicotinic ion channels noncompetitively, is able to reverse the neuromuscular paralysis after nerve agent poisoning in vitro and to protect guinea pigs against poisoning by nerve agents when used as part of a therapeutic drug combination including a muscarinic antagonist. In contrast to the oxime HI-6, this compound was equally effective in protecting against poisoning by sarin or tabun. Further studies should identify more effective compounds with this action and optimise doses for protection against nerve agent poisoning in vivo.
Assuntos
Substâncias para a Guerra Química/intoxicação , Antagonistas Nicotínicos/farmacologia , Compostos de Piridínio/farmacologia , Animais , Linhagem Celular Tumoral , Diafragma/efeitos dos fármacos , Diafragma/fisiologia , Relação Dose-Resposta a Droga , Cobaias , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Contração Muscular/efeitos dos fármacos , Antagonistas Nicotínicos/uso terapêutico , Intoxicação por Organofosfatos , Organofosfatos , Intoxicação/prevenção & controle , Compostos de Piridínio/uso terapêutico , Sarina/intoxicação , Soman/intoxicaçãoRESUMO
'Wash-out' and inactivation of the Ca current were examined in dialysed, voltage-clamped neurones of Helix aspersa under conditions that isolate the Ca current virtually free of other currents. EGTA or other internal Ca2+ chelators were routinely omitted from the dialysate. The time-dependent loss, or wash-out, of Ca current was slowed by addition to the dialysing solution of agents, such as dibutyryl adenosine 3'-5'-cyclic monophosphate (dibutyryl cyclic AMP), Mg adenosine 5'-triphosphate (ATP) and the catalytic subunit of cyclic-AMP-dependent protein kinase, that promote protein phosphorylation and by EGTA. However, neither the phosphorylation-promoting agents nor internal EGTA prevented wash-out entirely, nor did they significantly restore previously 'washed-out' current. With phosphorylating agents in the dialysing solution, the irreversible development of wash-out was greatly reduced by introduction of leupeptin, an inhibitor of protease activity. Thus, the irreversible component of wash-out appears to result from a Ca-dependent proteolytic process. In the presence of leupeptin alone, Ca current amplitude continued to decline: however, the current could be largely or fully restored with addition of catalytic subunit, dibutyryl cyclic AMP, and Mg ATP to the dialysing solution. Thus, inhibition of proteolysis revealed a reversible component of wash-out that appears to result from dephosphorylation. During perfusion with leupeptin, Mg ATP, dibutyryl cyclic AMP and catalytic subunit the Ca current remained stable for up to several hours without addition of internal Ca2+ buffer. The rate of inactivation of the current that occurs during a depolarizing step showed only a very gradual decline during this time. Under these conditions, perfusion with calcineurin, a Ca-calmodulin-dependent phosphatase, caused a significant increase in the rate of Ca current inactivation. This inactivation was virtually eliminated by introduction of EGTA or by replacement of external Ca2+ with Ba2+, which is consistent with the ion dependency for calmodulin-dependent activation of calcineurin. When ATP in the dialysate was replaced with ATP-gamma-S (adenosine 5'-O-(thiotriphosphate], an analogue that donates a thiophosphate group resistant to hydrolysis, the rate of inactivation slowed. Since Ca-dependent inactivation during step depolarizations is enhanced by conditions that promote dephosphorylation, and Ca current wash-out is slowed by conditions that promote phosphorylation, inactivation and reversible wash-out appear to be related.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Cálcio/fisiologia , Caracois Helix/fisiologia , Canais Iônicos/efeitos dos fármacos , Neurônios/fisiologia , Potenciais de Ação/efeitos dos fármacos , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Calmodulina/farmacologia , Proteínas de Ligação a Calmodulina/farmacologia , Cátions Bivalentes/farmacologia , Técnicas In Vitro , Neurônios/enzimologia , FosforilaçãoRESUMO
Computer-assisted modeling of calcium influx through voltage-activated membrane channels predicted that buffer-limited elevation of cytoplasmic free calcium ion concentration occurs within microscopic hemispherical "domains" centered upon the active Ca channels. With increasing depolarization, the number of activated channels, and hence the number of Ca domains, should increase; the single-channel current should, however, decrease, thereby decreasing Ca2+ accumulation in each domain relative to the macroscopic current. Such voltage dependence of the microscopic distribution of Ca2+ may influence relations between total Ca2+ entry and Ca-dependent processes. Ca-mediated inactivation of Ca channels in Aplysia neurons exhibits behavior consistent with the calcium domain hypothesis.
Assuntos
Cálcio/metabolismo , Canais Iônicos/metabolismo , Animais , Aplysia/metabolismo , Computadores , Eletroquímica , Cinética , Potenciais da Membrana , Modelos Biológicos , Neurônios/metabolismoRESUMO
Isolated neurons of Helix aspersa were dialyzed and voltage clamped under conditions that isolate the Ca current. The rapid time-dependent run-down, or washout, of Ca current could be slowed by addition of 1 mM EGTA to the dialysis solution. A more effective means of slowing washout was the use of agents that promote protein phosphorylation, such as cAMP, Mg-ATP and the catalytic subunit (CS) of cAMP-dependent protein kinase, along with leupeptin, a tripeptide inhibitor of proteases. In the presence of these agents, no internal EGTA was required to prevent Ca current washout. Thus, during dialysis with 100 microM leupeptin, 7 mM Mg-ATP and 20 micrograms/ml CS, the Ca current remained stable for up to several hours. The rate of Ca-dependent inactivation of the current that occurs during a depolarizing step showed only a small decline during prolonged dialysis. Under these conditions, introduction of 10 microM calmodulin plus 40 micrograms/ml calcineurin, a Ca-calmodulin-dependent phosphatase, caused a significant increase in the rate of Ca current inactivation during a depolarizing step. This increase in rate of inactivation, as well as the original inactivation, was eliminated by introduction of EGTA or replacement of external Ca with Ba, results that are consistent with the ion dependency for activation of calcineurin. When internal ATP was replaced with ATP-gamma-S, a hydrolysis-resistant analogue, the rate of Ca current inactivation slowed, providing further evidence that inactivation involves a dephosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cálcio/fisiologia , Neurônios/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Proteínas de Ligação a Calmodulina/metabolismo , Ácido Egtázico/farmacologia , Caracois Helix , Técnicas In Vitro , Canais Iônicos/fisiologia , Leupeptinas/farmacologia , Neurônios/efeitos dos fármacos , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/farmacologiaRESUMO
1. The effect of metabotropic glutamate receptor activation on intracellular Ca2+ activity (alpha Cai) of rat hippocampal pyramidal neurones in vitro was examined using ratiometric confocal laser scanning microscopy with the Ca(2+)-sensitive fluorescent probe indo-1 AM. 2. Metabotropic receptors were selectively activated with 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 100 microM) in the presence of D-2-amino 5-phosphonovaleric acid (D-APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and CdCl2. Most pyramidal neurones (77/84) responded with an elevation in Ca2+ activity, maximal after 3-5 min. Fluorescence ratio responses were concentration dependent (EC50 approximately 10 microM) and were blocked by prior application of the antagonist (RS)-4-carboxy-3-hydroxyphenylglycine (RS-CHPG, 300 microM). 3. Responses to 1S,3R-ACPD (100 microM) also caused acidification of the neurones, from estimated control pH 7.2 to pH 6.6 (measured with the pH-sensitive dye SNAFL-calcein). The correction factor for indo-1 determination of Ca2+ was estimated to be x 1.4. 4. Elevations in alpha Cai were greater within the perinuclear region (> 1000 nM), than in the cytoplasm (approximately 200 nM). This region was devoid of staining by the endoplasmic reticulum staining dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)). 5. It is concluded that activation of metabotropic receptors in immature rat hippocampal pyramidal neurones leads to a large increase in perinuclear Ca2+ which would be well positioned to interact with the genome.
Assuntos
Cálcio/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Cicloleucina/análogos & derivados , Cicloleucina/farmacologia , Citoplasma/metabolismo , Corantes Fluorescentes , Fura-2/análogos & derivados , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Microscopia Confocal , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Neurotoxinas/farmacologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Ratos , Ratos WistarRESUMO
Nicotinic acetylcholine receptors (nAChRs) are localised at morphologically distinct regions of the post-synaptic membrane by interactions between the receptor subunits and cytoskeletal proteins, such as the 43-kDa protein. We have used Xenopus oocytes to examine the localisation and pharmacological properties of muscle nAChRs associated with 43-kDa protein and to compare them with hybrid muscle nAChRs containing a beta subunit derived from a neuronal source. Receptors expressed on the oocyte outer membrane were visualised using confocal scanning laser microscopy. Coexpression of mouse muscle subunit alpha 1 beta 1 gamma delta and 43-kDa protein transcripts produced discrete receptor aggregates with a diameter of 1-5 microns whose function was partially blocked by application of neuronal bungarotoxin (NBT) at 100 nM. Substitution of the beta 1 subunit by the neuronal beta 2 protein produced a functioning receptor that did not aggregate in the presence of 43-kDa protein and was substantially blocked by the same concentration of NBT. Hybrid alpha 1 beta 4 gamma delta receptors exhibited a combination of characteristics in that they clustered like normal muscle subunits in the presence of 43-kDa protein, but showed a sensitivity to NBT intermediate between that of muscle receptors and that of hybrids containing beta 2. These results suggest that the beta subunit is an important determinant in receptor localisation and sensitivity to NBT.