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1.
Toxins (Basel) ; 14(4)2022 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-35448863

RESUMO

Gambierol inhibits voltage-gated K+ (KV) channels in various excitable and non-excitable cells. The purpose of this work was to study the effects of gambierol on single rat fetal (F19-F20) adrenomedullary cultured chromaffin cells. These excitable cells have different types of KV channels and release catecholamines. Perforated whole-cell voltage-clamp recordings revealed that gambierol (100 nM) blocked only a fraction of the total outward K+ current and slowed the kinetics of K+ current activation. The use of selective channel blockers disclosed that gambierol did not affect calcium-activated K+ (KCa) and ATP-sensitive K+ (KATP) channels. The gambierol concentration necessary to inhibit 50% of the K+ current-component sensitive to the polyether (IC50) was 5.8 nM. Simultaneous whole-cell current-clamp and single-cell amperometry recordings revealed that gambierol did not modify the membrane potential following 11s depolarizing current-steps, in both quiescent and active cells displaying repetitive firing of action potentials, and it did not increase the number of exocytotic catecholamine release events, with respect to controls. The subsequent addition of apamin and iberiotoxin, which selectively block the KCa channels, both depolarized the membrane and enhanced by 2.7 and 3.5-fold the exocytotic event frequency in quiescent and active cells, respectively. These results highlight the important modulatory role played by KCa channels in the control of exocytosis from fetal (F19-F20) adrenomedullary chromaffin cells.


Assuntos
Células Cromafins , Ciguatoxinas , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/farmacologia , Catecolaminas/farmacologia , Células Cultivadas , Ciguatoxinas/farmacologia , Potássio , Ratos
2.
Toxins (Basel) ; 12(8)2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32751635

RESUMO

The marine environment is known to be occupied by microorganisms. The potential toxicity of some of these marine microorganisms, that are capable of producing unknown biotoxins, has always been underestimated. Indeed, these biotoxins may be a threat to human health through the consumption of contaminated seafood and fish. For more than ten years, recurrent but atypical toxicity has been detected in mussels from Bizerte lagoon (North of Tunisia) during routine tests. In this study, we have isolated and characterized a new proteinaceous marine biotoxin, named Mussel Toxic Peptide (MTP). Using HPLC, electrophoresis and LC/MS studies, we showed that MTP has a protein characteristic UV-spectrum, can be visualized by protein specific reagents such as Coomassie, and has a molecular mass of 6.4 kDa. Patch-clamp experiments performed on cultured N18 neuroblastoma cells revealed that MTP (0.9-18 µM) markedly inhibited voltage-gated Na current, but was about 23 times less active in blocking voltage-gated K current at equimolar concentrations. To the best of our knowledge, this is the first time that a proteinaceous marine biotoxin with relatively high molecular mass is isolated and involved in the contamination of mussels harvested from shellfish farming areas.


Assuntos
Toxinas Marinhas , Mytilus , Animais , Linhagem Celular Tumoral , Estuários , Masculino , Toxinas Marinhas/química , Toxinas Marinhas/isolamento & purificação , Toxinas Marinhas/toxicidade , Camundongos Endogâmicos C57BL , Canais de Potássio/fisiologia , Canais de Sódio/fisiologia , Tunísia
3.
Neuroscience ; 439: 106-116, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31255710

RESUMO

Gambierol is a marine polycyclic ether toxin, first isolated from cultured Gambierdiscus toxicus dinoflagellates collected in French Polynesia. The chemical synthesis of gambierol permitted the analyses of its mode of action which includes the selective inhibition of voltage-gated K+ (KV) channels. In the present study we investigated the action of synthetic gambierol at vertebrate neuromuscular junctions using conventional techniques. Gambierol was studied on neuromuscular junctions in which muscle nicotinic ACh receptors have been blocked with d-tubocurarine (postsynaptic block), or in junctions in which quantal ACh release has been greatly reduced by a low Ca2+-high Mg2+ medium or by botulinum neurotoxin type-A (BoNT/A) (presynaptic block). Results show that nanomolar concentrations of gambierol inhibited the fast K+ current and prolonged the duration of the presynaptic action potential in motor nerve terminals, as revealed by presynaptic focal current recordings, increased stimulus-evoked quantal content in junctions blocked by high Mg2+-low Ca2+ medium, and by BoNT/A, reversed the postsynaptic block produced by d-tubocurarine and increased the transient Ca2+ signals in response to nerve-stimulation (1-10 Hz) in nerve terminals loaded with fluo-3/AM. The results suggest that gambierol, which on equimolar basis is more potent than 3,4-diaminopyridine, can have potential application in pathologies in which it is necessary to antagonize pre- or post-synaptic neuromuscular block, or both. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.


Assuntos
Ciguatoxinas , Potenciais de Ação , Animais , Junção Neuromuscular , Transmissão Sináptica , Vertebrados
4.
FEBS Lett ; 588(1): 35-40, 2014 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-24211835

RESUMO

Proteins from the oyster mushroom, 15 kDa ostreolysin A (OlyA), and 59 kDa pleurotolysin B (PlyB) with a membrane attack complex/perforin (MACPF) domain, damage cell membranes as a binary cytolytic pore-forming complex. Measurements of single-channel conductance and transmembrane macroscopic current reveal that OlyA/PlyB form non-selective ion-conducting pores with broad, skewed conductance distributions in N18 neuroblastoma and CHO-K1 cell membranes. Polyethylene-glycol 8000 (hydrodynamic radius of 3.78 nm) provides almost complete osmotic protection against haemolysis, which strongly suggests a colloid-osmotic type of erythrocyte lysis. Our data indicate that OlyA/PlyB form transmembrane pores of varied sizes, as other pore-forming proteins with a MACPF domain.


Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Proteínas Fúngicas/farmacologia , Proteínas Hemolisinas/farmacologia , Porinas/farmacologia , Animais , Células CHO , Bovinos , Linhagem Celular Tumoral , Membrana Celular/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Cricetinae , Cricetulus , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/fisiologia , Membrana Eritrocítica/ultraestrutura , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/ultraestrutura , Proteínas Fúngicas/metabolismo , Proteínas Hemolisinas/metabolismo , Hemólise/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Microscopia Eletrônica , Técnicas de Patch-Clamp , Pleurotus/metabolismo , Porinas/metabolismo
5.
Br J Pharmacol ; 166(5): 1654-68, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22229737

RESUMO

BACKGROUND AND PURPOSE: The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ-conopeptide (µ-CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant. EXPERIMENTAL APPROACH: µ-CnIIIC was sequenced, synthesized and characterized by its direct block of elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs and also on voltage-gated potassium channels and nicotinic acetylcholine receptors (nAChRs) to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data. KEY RESULTS: Synthetic µ-CnIIIC decreased twitch tension in mouse hemidiaphragms (IC(50) = 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), compared with µ-SIIIA, µ-SmIIIA and µ-PIIIA. µ-CnIIIC blocked Na(V)1.4 (IC(50) = 1.3 nM) and Na(V)1.2 channels in a long-lasting manner. Cardiac Na(V)1.5 and DRG-specific Na(V)1.8 channels were not blocked at 1 µM. µ-CnIIIC also blocked the α3ß2 nAChR subtype (IC(50) = 450 nM) and, to a lesser extent, on the α7 and α4ß2 subtypes. Structure determination of µ-CnIIIC revealed some similarities to α-conotoxins acting on nAChRs. CONCLUSION AND IMPLICATIONS: µ-CnIIIC potently blocked VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.


Assuntos
Conotoxinas/farmacologia , Caramujo Conus , Antagonistas Nicotínicos/farmacologia , Peptídeos/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Sequência de Aminoácidos , Animais , Conotoxinas/química , Esocidae , Feminino , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Camundongos , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Antagonistas Nicotínicos/química , Nervo Olfatório/efeitos dos fármacos , Nervo Olfatório/fisiologia , Oócitos , Peptídeos/química , Conformação Proteica , Receptores Nicotínicos/fisiologia , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/fisiologia , Bloqueadores dos Canais de Sódio/química , Canais de Sódio/fisiologia , Xenopus laevis
6.
Toxicon ; 56(5): 785-91, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20540957

RESUMO

Gambierol is a complex marine toxin first isolated with ciguatoxins from cell cultures of the toxic dinoflagellate Gambierdiscus toxicus. Despite the chemical complexity of the polycyclic ether toxin, the total successful synthesis of gambierol has been achieved by different chemical strategies. In the present work the effects of synthetic gambierol on mouse and frog skeletal neuromuscular preparations and Xenopus skeletal myocytes have been studied. Gambierol (0.1-5 muM) significantly increased isometric twitch tension in neuromuscular preparations stimulated through the motor nerve. Less twitch augmentation was observed in directly stimulated muscles when comparing twitch tension-time integrals obtained by nerve stimulation. Also, gambierol induced small spontaneous muscle contraction originating from presynaptic activity that was completely inhibited by d-tubocurarine. Gambierol slowed the rate of muscle action potential repolarization, triggered spontaneous and/or repetitive action potentials, and neither affected action potential amplitude nor overshoot in skeletal muscle fibers. These results suggest that gambierol through an action on voltage-gated K(+) channels prolongs the duration of action potentials, enhances the extent and time course of Ca(2+) release from the sarcoplasmic reticulum, and increases twitch tension generation. Further evidence is provided that gambierol at sub-micromolar concentrations blocks a fast inactivating outward K(+) current that is responsible for action potential prolongation in Xenopus skeletal myocytes.


Assuntos
Ciguatoxinas/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Feminino , Camundongos , Músculo Esquelético/fisiologia , Técnicas de Patch-Clamp , Tubocurarina/farmacologia , Xenopus
7.
Toxicon ; 56(5): 768-75, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19589350

RESUMO

The effects of Pacific ciguatoxin-4B (P-CTX-4B, also named gambiertoxin), extracted from toxic Gambierdiscus dinoflagellates, were assessed on nodal K(+) and Na(+) currents of frog myelinated axons, using a conventional voltage-clamp technique. P-CTX-4B decreased, within a few minutes, both K(+) and Na(+) currents in a dose-dependent manner, without inducing any marked change in current kinetics. The toxin was more effective in blocking K(+) than Na(+) channels. P-CTX-4B shifted the voltage-dependence of Na(+) conductance by about 14 mV towards more negative membrane potentials. This effect was reversed by increasing Ca(2+) in the external solution. A negative shift of about 16 mV in the steady-state Na(+) inactivation-voltage curve was also observed in the presence of the toxin. Unmodified and P-CTX-4B-modified Na(+) currents were similarly affected by the local anaesthetic lidocaine. The decrease of the two currents by lidocaine was dependent on both the concentration and the membrane potential during pre-pulses. In conclusion, P-CTX-4B appears about four times more effective than P-CTX-1B to affect K(+) channels, whereas it is about 50 times less efficient to affect Na(+) channels of axonal membranes. These actions may be related to subtle differences between the two chemical structures of molecules.


Assuntos
Axônios/efeitos dos fármacos , Ciguatoxinas/toxicidade , Dinoflagellida/química , Bainha de Mielina/metabolismo , Canais de Potássio/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacos , Animais , Masculino , Rana esculenta
8.
Toxicon ; 56(5): 759-67, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19646468

RESUMO

Caribbean ciguatoxin-1 (C-CTX-1) induced, after about 1h exposure, muscle membrane depolarisation and repetitive post-synaptic action potentials (APs) in frog neuromuscular preparations. This depolarising effect was also observed in a Ca(2+)-free medium with a strong enhancement of spontaneous quantal transmitter release, compared with control conditions. The ciguatoxin-induced increase in release could be accelerated when Ca(2+) was present in the extracellular medium. C-CTX-1 also enhanced nerve-evoked quantal acetylcholine (ACh) release. At normal neuromuscular junctions loaded with the fluorescent dye FM1-43, C-CTX-1 induced swelling of nerve terminals, an effect that was reversed by hyperosmotic d-mannitol. In myelinated axons, C-CTX-1 increased nodal membrane excitability, inducing spontaneous and repetitive APs. Also, the toxin enlarged the repolarising phase of APs in control and tetraethylammonium-treated axons. Overall, our data suggest that C-CTX-1 affects nerve excitability and neurotransmitter release at nerve terminals. We conclude that C-CTX-1-induced up-regulation of Na(+) channels and the inhibition of K(+) channels, at low nanomolar concentrations, produce a variety of functional dysfunctions that are in part responsible for the human muscle skeletal symptoms observed in ciguatera. All these dysfunctions seem to result from the subtle balance between ionic currents, intracellular Na(+) and Ca(2+) concentrations, and engaged second messengers.


Assuntos
Axônios/efeitos dos fármacos , Ciguatoxinas/toxicidade , Bainha de Mielina/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Região do Caribe , Ciguatoxinas/química , Estrutura Molecular , Rana esculenta
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