RESUMO
The autofluorescence of biological tissue can be exploited for the detection and diagnosis of disease but, to date, its complex nature and relatively weak signal levels have impeded its widespread application in biology and medicine. We present here a portable instrument designed for the in situ simultaneous measurement of autofluorescence emission spectra and temporal decay profiles, permitting the analysis of complex fluorescence signals. This hyperspectral fluorescence lifetime probe utilizes two ultrafast lasers operating at 355 and 440 nm that can excite autofluorescence from many different biomolecules present in skin tissue including keratin, collagen, nicotinamide adenine dinucleotide (phosphate), and flavins. The instrument incorporates an optical fiber probe to provide sample illumination and fluorescence collection over a millimeter-sized area. We present a description of the system, including spectral and temporal characterizations, and report the preliminary application of this instrument to a study of recently resected (<2 h) ex vivo skin lesions, illustrating its potential for skin cancer detection and diagnosis.
Assuntos
Biomarcadores Tumorais/análise , Medições Luminescentes/instrumentação , Neoplasias Cutâneas/diagnóstico , Espectrometria de Fluorescência/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Técnicas de Sonda Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodosRESUMO
Amyloid beta-peptide (Abeta) is the main component of senile plaques which characterize Alzheimer's disease and may induce neuronal death through mechanisms which include oxidative stress. To date, the signalling pathways linking oxidant stress, a component of several neurodegenerative diseases, to cell death in the CNS are poorly understood. Melastatin-like transient receptor potential 2 (TRPM2) is a Ca(2+)-permeant non-selective cation channel, which responds to increases in oxidative stress levels in the cell and is activated by oxidants such as hydrogen peroxide. We demonstrate here that Abeta and hydrogen peroxide both induce death in cultured rat striatal cells which express TRPM2 endogenously. Transfection with a splice variant that acts as a dominant negative blocker of TRPM2 function (TRPM2-S) inhibited both hydrogen peroxide- and Abeta-induced increases in intracellular-free Ca(2+) and cell death. Functional inhibition of TRPM2 activation by the poly(ADP-ribose)polymerase inhibitor SB-750139, a modulator of intracellular pathways activating TRPM2, attenuated hydrogen peroxide- and Abeta-induced cell death. Furthermore, a small interfering RNA which targets TRPM2, reduced TRPM2 mRNA levels and the toxicity induced by hydrogen peroxide and Abeta. These data demonstrate that activation of TRPM2, functionally expressed in primary cultures of rat striatum, contributes to Abeta- and oxidative stress-induced striatal cell death.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Clusterina/metabolismo , Peróxido de Hidrogênio/toxicidade , Neurônios/metabolismo , Oxidantes/toxicidade , Fragmentos de Peptídeos/toxicidade , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Clusterina/genética , Corpo Estriado/citologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Like a number of other TRP channels, TRPM2 is a Ca(2+)-permeable non-selective cation channel, the activity of which is regulated by intracellular and extracellular Ca(2+). A unique feature of TRPM2 is its activation by ADP-ribose and chemical species that arise during oxidative stress, for example, NAD(+) and H(2)O(2). These properties have lead to proposals that this channel may play a role in the cell death produced by pathological redox states. The lack of known antagonists of this channel have made these hypotheses difficult to test. Here, we demonstrate, using patch clamp electrophysiology, that the non-steroidal anti-inflammatory compound flufenamic acid (FFA) inhibits recombinant human TRPM2 (hTRPM2) as well as currents activated by intracellular ADP-ribose in the CRI-G1 rat insulinoma cell line. All concentrations tested in a range from 50 to 1000 microM produced complete inhibition of the TRPM2-mediated current. Following FFA removal, a small (typically 10-15%) component of current was rapidly recovered (time constant approximately 3 s), considerably longer periods in the absence of FFA produced no further current recovery. Reapplication of FFA re-antagonised the recovered current and subsequent FFA washout produced recovery of only a small percentage of the reblocked current. Decreasing extracellular pH accelerated FFA inhibition of TRPM2. Additional experiments indicated hTRPM2 activation was required for FFA antagonism to occur and that the generation of irreversible antagonism was preceded by a reversible component of block. FFA inhibition could not be induced by intracellular application of FFA. ADP-ribose activated currents in the rat insulinoma cell line CRI-G1 were also antagonised by FFA with concentration- and pH-dependent kinetics. In contrast to the observations made with hTRPM2, antagonism of ADP-ribose activated currents in CRI-G1 cells could be fully reversed following FFA removal. These experiments suggest that FFA may be a useful tool antagonist for studies of TRPM2 function.
Assuntos
Ácido Flufenâmico/farmacologia , Concentração de Íons de Hidrogênio , Canais Iônicos/antagonistas & inibidores , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Adenosina Difosfato Ribose/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Humanos , Insulinoma , Canais Iônicos/química , Canais Iônicos/fisiologia , Rim , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos , Proteínas Recombinantes/farmacologia , Canais de Cátion TRPMRESUMO
The emergence of the TRP (C) and vanilloid (TRPV) receptor family of Ca(2+) permeable channels has started to provide molecular focus to a linked group of ion channels whose common feature is activation primarily by intracellular ligands. These channels have a central role in Ca(2+) homeostasis in virtually all cells and in particular those that lack voltage-gated Ca(2+) channels. We will discuss recent work that is more precisely defining both molecular form and physiological function of this important group of Ca(2+) permeable channels with particular focus on the intracellular ligands that gate and modulate channel activity.
Assuntos
Canais de Cálcio/fisiologia , Ativação do Canal Iônico/fisiologia , Lipídeos/fisiologia , Receptores de Droga/fisiologia , Animais , Canais de Cálcio/genética , Humanos , Receptores de Droga/genética , Canais de Cátion TRPC , Transcrição GênicaRESUMO
Electrophysiological recordings from identified noradrenergic locus coeruleus (LC) neurones in rat brain slices have revealed that the orexins can cause direct and reversible depolarisation of the postsynaptic membrane. Whilst it is known that the membrane depolarisation produced by orexin-A can triple the firing rate of spontaneously active LC neurones, quantitative pharmacological analysis that determines the receptor subtype(s) mediating the orexinergic response has not yet been performed. Here we demonstrate that the effects of orexin-A are five-fold more potent than orexin-B on LC neuronal excitability. We show further that the orexin receptor antagonist SB-334867-A inhibits the effects of both agonists with pK(B) values similar to those calculated for human OX1 receptors expressed in CHO cells. Finally, we found no evidence for tonic activation of OX1 receptors in LC noradrenergic neurones despite electron microscopic evidence that orexin terminals directly contact these neurones. These data demonstrate that SB-334867-A is a useful tool compound with which to study the physiology of OX1 receptors.
Assuntos
Benzoxazóis/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular , Locus Cerúleo/efeitos dos fármacos , Neuropeptídeos/antagonistas & inibidores , Ureia/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Proteínas de Transporte/farmacologia , Eletrofisiologia , Técnicas In Vitro , Masculino , Naftiridinas , Neuropeptídeos/farmacologia , Receptores de Orexina , Orexinas , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/metabolismo , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/metabolismo , Receptores de Neuropeptídeos , Ureia/análogos & derivadosAssuntos
Anestésicos Locais/farmacologia , Dor/fisiopatologia , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/genética , Tetrodotoxina/farmacologia , Animais , Animais Geneticamente Modificados , Resistência a Medicamentos , Humanos , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , Dor/tratamento farmacológicoRESUMO
The effects of energy substrate removal and metabolic pathway block have been examined on neuronal and glial survival in organotypic slice cultures of rat hippocampus. Slice cultures resisted 24 h of exogenous energy substrate deprivation. Application of 0.5 mM alpha-cyano-4-hydroxycinnamate (4-CIN) for 24 h resulted in specific damage to neuronal cell layers, which could be reversed by co-application of 5 mM lactate. Addition of 10 mM 2-deoxyglucose in the absence of exogenous energy supply produced widespread cell death throughout the slice. This was partly reversed by co-application of 5 mM lactate. These effects of metabolic blockade on cell survival were qualitatively similar to the effects on population spikes recorded in the CA1 cell layer following 60 min application of these agents. The data suggest that monocarboxylate trafficking from glia to neurons is an essential route for supply of energy substrates to neurons particularly when exogenous energy supply is restricted.
Assuntos
Proteínas de Transporte/fisiologia , Glucose/deficiência , Hipocampo/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Proteínas de Transporte de Ânions , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ácidos Cumáricos/farmacologia , Desoxiglucose/farmacologia , Combinação de Medicamentos , Eletrofisiologia , Metabolismo Energético , Hipocampo/citologia , Técnicas In Vitro , Ácido Láctico/farmacologia , Neuroglia/fisiologia , Neurônios/fisiologia , Ratos , Ratos WistarRESUMO
For progression to clinical trials in stroke, putative neuroprotective compounds should show robust efficacy post-ischaemia in several experimental models of stroke. This paper describes the characterisation of (+)(1S, 2R)-cis-1-[4-(1-methyl-1-phenylethyl)phenoxy]-2-methylamino indane hydrochloride (SB-221420-A), a Ca(2+) and Na(+) channel antagonist. SB-221420-A inhibited (IC(50)=2.2 microM) N-type voltage-operated Ca(2+) channel currents in cultured superior cervical ganglion neurons, which were pretreated with 10 microM nimodipine to block L-type voltage-operated Ca(2+) channel currents. In dorsal root ganglion neurons pretreated with 1 microM omega-conotoxin GVIA to block N-type voltage-operated Ca(2+) channel currents, SB-221420-A inhibited the residual Ca(2+) current with an IC(50) of 7 microM. SB-221420-A also inhibited Na(+) currents in dorsal root ganglion neurons with an IC(50) of 8 microM. In rats, the pharmacokinetic profile of SB-221420-A shows that it has a half-life of 6.4 h, a high volume of distribution, is highly brain penetrating, and has no persistent metabolites. Following bilateral carotid artery occlusion in gerbils, SB-221420-A significantly reduced the level of ischaemia-induced hyperlocomotor activity and the extent of hippocampal CA1 cell loss compared to the ischaemic vehicle-treated group. SB-221420-A was also effective in focal models of ischaemia. In the mouse permanent middle cerebral artery occlusion model, SB-221420-A (10 mg/kg) administered intravenously, post-ischaemia significantly (P<0.05) reduced lesion volume compared to the ischaemic vehicle-treated group. In the normotensive rat permanent middle cerebral artery occlusion model, SB-221420-A (10 mg/kg) administered intravenously over 1 h, beginning 30 min postmiddle cerebral artery occlusion, significantly (P<0.05) reduced lesion volume from 291+/-16 to 153+/-30 mm(3), compared to ischaemic vehicle-treated controls when measured 24 h postmiddle cerebral artery occlusion. Efficacy was maintained when the same total dose of SB-221420-A was infused over a 6-h period, beginning 30 min postmiddle cerebral artery occlusion. SB-221420-A also significantly (P<0.05) reduced lesion volume following transient middle cerebral artery occlusion in normotensive rats and permanent middle cerebral artery occlusion in spontaneously hypertensive rats (SHR). Investigation of the side effect profile using the Irwin screen in mice revealed that, at neuroprotective doses, there were no overt behavioural or cardiovascular changes. These data demonstrate that robust neuroprotection can be seen post-ischaemia with SB-221420-A in both global and focal ischaemia with no adverse effects at neuroprotective doses, and indicate the potential utility of a mixed cation blocker to improve outcome in cerebral ischaemia.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Indanos/farmacologia , Fármacos Neuroprotetores/farmacologia , Bloqueadores dos Canais de Sódio , Acidente Vascular Cerebral/prevenção & controle , Anestesia , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Estenose das Carótidas/fisiopatologia , Estenose das Carótidas/prevenção & controle , Células Cultivadas , Estado de Consciência , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Gerbillinae , Hemodinâmica/efeitos dos fármacos , Hipertensão/fisiopatologia , Indanos/farmacocinética , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/prevenção & controle , Ataque Isquêmico Transitório/fisiopatologia , Ataque Isquêmico Transitório/prevenção & controle , Masculino , Potenciais da Membrana/efeitos dos fármacos , Taxa de Depuração Metabólica , Camundongos , Atividade Motora/efeitos dos fármacos , Neurônios Aferentes/citologia , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Acidente Vascular Cerebral/fisiopatologia , Distribuição TecidualRESUMO
We have explored the neuroprotective efficacy of the cell penetrant caspase inhibitor, Ac-YVAD-cmk, in a hippocampal slice model of neuronal cell death induced by oxygen and glucose deprivation. Organotypic hippocampal slice cultures were prepared from 8 to 10-day-old rats and maintained for 10 to 12 days in vitro. Pre-treatment with Ac-YVAD-cmk prior to 45 min oxygen and glucose deprivation was neuroprotective as measured by propidium iodide uptake, with an EC(50) between 1 and 10 micromol/l. Ac-YVAD-cmk was also able to preserve synaptic function in the organotypic hippocampal slice cultures 24 h after oxygen and glucose deprivation. Ac-YVAD-cmk prevented the increase in histone-associated DNA fragmentation induced by oxygen and glucose deprivation. Interleukin-1beta did not reverse the protective effect of Ac-YVAD-cmk, and interleukin-1 receptor antagonist alone was not protective. These results show that caspase inhibitors are neuroprotective in a hippocampal slice culture system, using structural, biochemical and electrophysiological endpoints, and that this effect is not a result of inhibition of interleukin-1beta production.
Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase , Morte Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Neurônios/citologia , Fármacos Neuroprotetores/farmacologia , Animais , Isquemia Encefálica/tratamento farmacológico , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Transformada , Fragmentação do DNA/efeitos dos fármacos , Eletrofisiologia , Glucose/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Oxigênio/farmacologia , Ratos , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
We have cloned human TREK-1, one of the newly emerging mammalian family of 2-P domain potassium channels. The channel has 411 amino acids with a 41-amino-acid extension at the C-terminus when compared with the cloned mouse TREK-1 channel. Expression of hTREK-1 produced a substantial hyperpolarising shift in resting membrane potential accompanied by the induction of large, outwardly rectifying, non-inactivating currents which were potassium selective. Pharmacologically, hTREK-1-mediated currents were only blocked to a limited extent by classic potassium channel blockers or open channel pore blockers known to potently inhibit other channels. The channel was reversibly potentiated by arachidonic acid. CNS distribution of hTREK-1 is widespread with higher levels being observed in caudate, putamen, amygdala, thalamus and spinal cord. Only low levels of expression were seen in the majority of peripheral regions. Thus, hTREK-1, although functionally and pharmacologically similar to mouse TREK-1, appears to have a more CNS-specific distribution.
Assuntos
Clonagem Molecular , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/genética , Canais de Potássio/metabolismo , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Linhagem Celular , Humanos , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Distribuição Tecidual , Xenopus laevis/metabolismoRESUMO
1. Effects of external pH on the human P2X4 purinoceptor, an ATP-activated ion channel, were studied using the Xenopus oocyte expression system. 2. Changing the external pH from 7.4 to 6.5 significantly reduced, whilst an increase to pH 8 enhanced, maximum ATP-activated current amplitude, without changing the current- voltage relationship of the ATP-activated current. 3. Diethyl pyrocarbonate (DEPC; 10 mM) treatment of P2X4-injected oocytes had no effect on the pH sensitivity of the ATP-activated current. 4. Site-directed mutagenesis of histidine 286 (H286) to alanine completely abolished the pH sensitivity of the P2X4 receptor at all agonist concentrations. ATP potency showed a small (fourfold) leftward shift. Mutagenesis of the other three histidines present in the P2X4 sequence had no effect on pH sensitivity. 5. The results show that pH modulation of P2X4 in the pathophysiological range is mediated by protonation of H286. This provides direct confirmation that pH sensitivity resides in the P2X4 channel protein rather than the agonist species.
Assuntos
Espaço Extracelular/metabolismo , Hidrogênio/metabolismo , Mutação/fisiologia , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/fisiologia , Animais , Dietil Pirocarbonato/farmacologia , Condutividade Elétrica , Feminino , Humanos , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Oócitos , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2X4 , Xenopus laevisRESUMO
The inhibitory effects of the omega-conotoxins GVIA, MVIIA and MVIIC on electrically-evoked, tetrodotoxin (10(-7) M)-sensitive, autonomic nerve activity were studied using human, rat or guinea-pig vas deferens and intestinal tissues. In each preparation from each species, nM concentrations of omega-conotoxins GVIA and MVIIA prevented the neuronally-mediated contractions, whereas omega-conotoxin MVIIC was either markedly less potent (IC(50)'s 1.4 or 2.9 log units more than for omega-conotoxin GVIA in guinea-pig ileum and rat vas deferens, respectively) or was without significant activity (human vas deferens, human Taenia coli) when tested at similar concentrations. In contrast the differences in potency between omega-conotoxins GVIA and MVIIC were considerably less when assayed directly on Ca(2+) channel currents evoked from rat superior cervical ganglion neurons in culture (approximately 0.1 log unit difference) and from a stable cell line expressing rat alpha(1B), alpha(2)delta, beta(1b) Ca(2+) channel subunits (approximately 0.9 log unit). These different rank-orders of inhibitory activity of the conotoxins support the suggestion that there are pharmacologically distinct N-type Ca(2+) channels in the peripheral nervous system, and that this tissue-dependent difference is seen in man.
Assuntos
Sistema Nervoso Autônomo/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/efeitos dos fármacos , ômega-Conotoxinas/farmacologia , Animais , Colo/efeitos dos fármacos , Colo/inervação , Estimulação Elétrica , Gânglios Parassimpáticos/efeitos dos fármacos , Cobaias , Humanos , Íleo/efeitos dos fármacos , Íleo/inervação , Técnicas In Vitro , Masculino , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Junção Neuroefetora/efeitos dos fármacos , Ratos , Tetrodotoxina/farmacologia , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/inervação , ômega-Conotoxina GVIA/farmacologiaRESUMO
The localization of orexin neuropeptides in the lateral hypothalamus has focused interest on their role in ingestion. The orexigenic neurones in the lateral hypothalamus, however, project widely in the brain, and thus the physiological role of orexins is likely to be complex. Here we describe an investigation of the action of orexin A in modulating the arousal state of rats by using a combination of tissue localization and electrophysiological and behavioral techniques. We show that the brain region receiving the densest innervation from orexinergic nerves is the locus coeruleus, a key modulator of attentional state, where application of orexin A increases cell firing of intrinsic noradrenergic neurones. Orexin A increases arousal and locomotor activity and modulates neuroendocrine function. The data suggest that orexin A plays an important role in orchestrating the sleep-wake cycle.
Assuntos
Nível de Alerta/fisiologia , Proteínas de Transporte/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Locus Cerúleo/fisiologia , Neuropeptídeos/fisiologia , Animais , Comportamento Animal/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Corticosterona/metabolismo , Relação Dose-Resposta a Droga , Eletroencefalografia , Eletrofisiologia , Hormônio do Crescimento/metabolismo , Imuno-Histoquímica , Masculino , Aprendizagem em Labirinto/fisiologia , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Orexinas , Prolactina/metabolismo , Ratos , Ratos Sprague-Dawley , Sono/fisiologia , Fatores de TempoRESUMO
The novel anticonvulsant SB-204269 inhibits epileptiform afterdischarges induced by high K+ in rat hippocampal slices. Its effects on voltage-gated Na+ currents, measured from cultured hippocampal neurones using whole cell patch clamp, were compared to the effects of existing anticonvulsants. SB-204269 produced no significant tonic block of Na+ currents nor any voltage-dependent and frequency-dependent block at doses 50 to 500 fold higher than its anticonvulsant EC50 of 0.2 microM. In contrast, lamotrigine, phenytoin and carbamazepine at 50 microM, blocked Na+ currents in a voltage-dependent manner. SB-204269 also had no effect on action potential discharges evoked by elevating external K+. These data suggest that direct blockade of voltage-gated channels does not contribute to the anticonvulsant properties of SB-204269 and further support the hypothesis that this compound has a novel mechanism of action.
Assuntos
Anticonvulsivantes/farmacologia , Benzamidas/farmacologia , Benzopiranos/farmacologia , Hipocampo/fisiologia , Neurônios/fisiologia , Canais de Sódio/fisiologia , Animais , Carbamazepina/farmacologia , Células Cultivadas , Embrião de Mamíferos , Hipocampo/citologia , Lamotrigina , Potenciais da Membrana/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Fenitoína/farmacologia , Ratos , Bloqueadores dos Canais de Sódio , Triazinas/farmacologiaAssuntos
Canais de Cálcio/genética , Peptídeos/farmacologia , Processamento Alternativo , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , DNA Complementar/genética , Microinjeções , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ratos , Xenopus , ômega-Conotoxina GVIARESUMO
It has been suggested that, after ischaemia, activation of proteases such as calpains could be involved in cytoskeletal degradation leading to neuronal cell death. In vivo, calpain inhibitors at high doses have been shown to reduce ischaemic damage and traumatic brain injury, however, the relationship between calpain activation and cell death remains unclear. We have investigated the role of calpain activation in a model of ischaemia based on organotypic hippocampal slice cultures using the appearance of spectrin breakdown products (BDPs) as a measure of calpain I activation. Calpain I activity was detected on Western blot immediately after a 1-h exposure to ischaemia. Up to 4 h post ischaemia, BDPs were found mainly in the CA1 region and appeared before uptake of the vital dye propidium iodide (PI). 24 h after the insult, BDPs were detected extensively in CA1 and CA3 pyramidal cells, all of which was PI-positive. However, there were many more PI-positive cells that did not have BDPs, indicating that the appearance of BDPs does not necessarily accompany ischaemic cell death. Inhibition of BDP formation by the broad-spectrum protease inhibitor leupeptin was not accompanied by any neuroprotective effects. The more specific and more cell-permeant calpain inhibitor MDL 28170 had a clear neuroprotective effect when added after the ischaemic insult. In contrast, when MDL 28170 was present throughout the entire pre- and post-incubation phases, PI labelling actually increased, indicating a toxic effect. These results suggest that calpain activation is not always associated with cell death and that, while inhibition of calpains can be neuroprotective under some conditions, it may not always lead to beneficial outcomes in ischaemia.
Assuntos
Calpaína/antagonistas & inibidores , Calpaína/fisiologia , Glucose/deficiência , Hipocampo/metabolismo , Hipóxia/metabolismo , Animais , Corantes , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipóxia/patologia , Imuno-Histoquímica/métodos , Técnicas In Vitro , Propídio , Ratos , Ratos Wistar , Espectrina/antagonistas & inibidores , Espectrina/metabolismo , Coloração e RotulagemRESUMO
In response to extracellular application of 50 microM ATP, all individual porcine aortic smooth muscle cells respond with rapid rises from basal [Ca2+]i to peak [Ca2+]i within 5 s. The time from stimulus to the peak of the [Ca2+]i response increases with decreasing concentration of ATP. At ATP concentrations of 0.5 microM and below, the time to the [Ca2+]i peak varies more significantly from cell to cell than at higher concentrations, and each cell shows complicated initiation and decay kinetics. For any individual cell, the lag phase before a response decreases with increasing concentration of ATP. An increase in lag time with decreasing ATP concentration is also observed in the absence of extracellular Ca2+, but the lag phase is more pronounced, especially at concentrations of ATP below 0.5 microM. Whole-cell patch-clamp electrophysiology shows that in porcine aortic smooth muscle cells, ATP stimulates an inward current carried mainly by Cl- ion efflux with a time course similar to the [Ca2+]i changes and no detectable current from an ATP-gated cation channel. A simple signal cascade initiation kinetics model, starting with nucleotide receptor activation leading to IP3-mediated Ca2+ release from IP3-sensitive internal stores, fits the data and suggests that the kinetics of the Ca2+ response are dominated by upstream signal cascade components.
Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Túnica Média/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Aorta , Cálcio/farmacologia , Células Cultivadas , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Suínos , Fatores de Tempo , Túnica Média/citologia , Túnica Média/efeitos dos fármacosRESUMO
1. SB-205384, and its (+) enantiomer (+)-SB-205384 were tested for their modulatory effects on human GABA(A) receptor subunit combinations expressed in Xenopus oocytes by electrophysiological methods. 2. The slowing of the decay rate induced by SB-205384 on native GABA-activated currents in rat neurones was also seen on GABA(A) currents in oocytes expressing human GABA(A) subunits. This temporal effect was observed for the alpha3beta2gamma2 subunit combination with little effect in subunit combinations containing either alpha1 or alpha2. 3. Potentiation of the peak amplitude of the GABA-activated currents by SB-205384 or (+)-SB-205384 was less specific for a particular subunit combination, although the greatest effect at 10 microM drug was seen on the alpha3beta2gamma2 subunit combination. 4. In contrast, zolpidem, a benzodiazepine site modulator, did not significantly slow decay rates of GABA(A) currents in oocytes expressing the alpha3beta2gamma2 subunit combination. Zolpidem, as expected, did selectively potentiate GABA-activated currents on oocytes expressing the gamma2 subunit compared to those containing the gamma1. 5. The results show that the novel kinetic modulatory profile of SB-205384 is selective for the alpha3beta2gamma2 subunit combination. This suggests that the compound is binding to a novel regulatory site on the subunit complex.
Assuntos
Aminopiridinas/farmacologia , Moduladores GABAérgicos/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Tiofenos/farmacologia , Animais , Feminino , Humanos , Piridinas/farmacologia , Ratos , Receptores de GABA-A/genética , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Xenopus laevis , ZolpidemRESUMO
1. 4-Amino-7-hydroxy-2-methyl-5,6,7,8,-tetrahydrobenzo[b]thieno[2,3-b]pyrid ine-3-carboxylic acid, but-2-ynyl ester (SB-205384) and other gamma-aminobutyric acid(A) (GABA(A)) receptor modulators were tested for their effects on GABA-activated chloride currents in rat cerebellar granule cells by use of the whole-cell patch clamp technique. 2. The major effect of SB-205384 on GABA(A)-activated current was an increase in the half-life of decay of the response once the agonist had been removed. This is in contrast to many GABA(A) receptor modulators that have previously been shown to potentiate GABA-activated currents. 3. This profile could be explained if SB-205384 stabilizes the channel in open and desensitized states so that channel closing is dramatically slowed. Such a modulatory profile may produce a novel behavioural profile in vivo.