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J Molgó

Publications and source records attributed to J Molgó.

At least 19 recordsLinked to original sources
PubMed2000

Recycling and refilling of transmitter quanta at the frog neuromuscular junction.

1. Fluorescent dyes have been used at the frog neuromuscular junction to label synaptic vesicular membrane. Retrieved membrane is reformed into vesicles, which are released along with pre-existing vesicles. Consequently, if vesicular refilling with acetylcholine (ACh) is depressed by inhibitors, two sizes of quanta should be released: normal and smaller. As recycling continues the fraction of smaller size quanta should increase exponentially. 2. We enhanced the rate of quantal release by elevating the K+ concentration. The principal inhibitors were (-)-vesamicol (VES), hemicholinium-3 (HC3), and NH4+. Quantal size measurements were fitted to one and to two cumulative lognormal probability distribution functions. When two fitted better, the statistical significance assessment took into account the three additional parameters used in calculating the fit. 3. After recycling in the presence of inhibitor, many sets were fitted better by two lognormal functions. As recycling continued, the fraction of the miniature endplate potential voltage-time integrals ( MEPPs) in the larger sub-population decreased exponentially. 4. The size of the releasable pool was estimated by counting the quanta released by carbonyl cyanide m-chlorophenylhydrazone (CCCP). This was compared to pool sizes calculated from the inhibitor experiments. The two estimates of pool size were indistinguishable, with mean values ranging from about 170,000 to 270,000. 5. With all of the treatments tested, the means of the sizes in the smaller sub-population of MEPPs were about 1/3 those of the larger sub-populations. 6. Recycling synaptic vesicles appear to be incorporated into the releasable pool from which they have roughly the same probability of release as the pre-existing vesicles.

Animals↗
PubMed2000

Ca(2+) and Na(+) contribute to the swelling of differentiated neuroblastoma cells induced by equinatoxin-II.

Equinatoxin-II (EqTx-II), a cytotoxic protein (mol.wt 20 kDa) isolated from the sea anemone Actinia equina, was found to consistently increase the three-dimensional projected area of differentiated neuroblastoma (NG108-15) cells provided Ca(2+) was present in the medium. No swelling was detected when external NaCl was replaced by sucrose, but replacement of NaCl by Na-isethionate did not prevent the swelling, as revealed by confocal laser scanning microscopy. In addition, microspectrofluorometric measurements in cells preloaded with the Ca(2+) indicator fura-2/AM revealed that EqTx-II (100 nM) markedly increased the fluorescence (F(340)/F(380)) ratio indicating a rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). The elevation of [Ca(2+)](i) exhibited two components that seem to be related to the kinetics of EqTx-II-induced Ca(2+) entry since pretreatment of cells with Ca(2+)-ATPase inhibitors (thapsigargin), Ca(2+) channel blockers (nifedipine and Gd(3+)) or prolonged exposure to a high K(+) (75 mM) medium did not alter EqTx-II-induced Ca(2+) signals. As far as we know, this is the first demonstration that EqTx-II causes swelling of neuroblastoma cells and that this effect is correlated both with an increase of [Ca(2+)](i) and needs the presence of extracellular Na(+). It is suggested that EqTx-II has the ability to insert into the plasma membrane of neuroblastoma cells and to form pores altering the membrane permeability and the intracellular osmolality, inducing a marked influx of water into the cells.

Animals↗
PubMed2000

Effects of trachynilysin, a protein isolated from stonefish (Synanceia trachynis) venom, on frog atrial heart muscle.

The effects of trachynilysin (TLY), a protein toxin isolated from stonefish (Synanceia trachynis) venom, were studied on the electrical and mechanical activities of frog atrial fibres. TLY (1 microg/ml) hyperpolarized the membrane, shortened the action potential (AP) duration (APD), exerted a negative inotropic effect and elicited contracture. These effects did not develop in the presence of atropine. TLY shortened the APD of fibres isolated from a frog completely paralyzed with botulinum type A toxin, in the presence of Ca2+ but not when Ca2+ was replaced by Sr2+. TLY increased the basal and the peak of the fluorescence ratio of stimulated fibres loaded with fura-2. Confocal laser scanning microscopy revealed the existence of a diffuse innervation in atrial tissue. Our results suggest that TLY enhances the release of acetylcholine from atrial cholinergic nerve terminals and activates indirectly muscarinic receptors leading to a shortening of APD. They also show that the mechanical effects induced by TLY are due to an increase of the Ca2+ influx and to a rise in intracellular Ca2+ levels which leads to (i) a slowing of the Na+/Ca2+ exchange activity, which accounts for the contracture and (ii) the activation of a Ca2+-dependent K+ current involved in the APD shortening.

Acetylcholine↗
PubMed2000

Regulated expression of the proteoglycan SPOCK in the neuromuscular system.

SPOCK is prevalent in developing synaptic fields of the central nervous system (Charbonnier et al., 2000. Mech. Dev. 90, 317-321). The expression of SPOCK during neuromuscular junction (NMJ) formation was compared to agrin and acetylcholine receptor (AChR) distribution. SPOCK is detected within the myogenic masses during the early steps of embryonic development, and distributed in the cytoplasm of myotubes before coclustering with AChRs. In the adult, SPOCK is present in axons and is highly expressed by Schwann cells. SPOCK altered expression pattern after nerve lesioning, or cholinergic transmission blockade, strongly indicate that its cellular distribution at the NMJ depends on innervation.

Animals↗
PubMed2000

Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+.

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.

Animals↗
PubMed1999

Hyperosmolar D-mannitol reverses the increased membrane excitability and the nodal swelling caused by Caribbean ciguatoxin-1 in single frog myelinated axons.

The effects of hyperosmolar D-mannitol were studied on single frog myelinated nerve fibres previously poisoned with Caribbean ciguatoxin-1 (C-CTX-1), a new toxin isolated from the pelagic fish Caranx latus inhabiting the Caribbean region. In current-clamped myelinated axons, C-CTX-1 (50-120 nM) caused spontaneous and repetitive action potential discharges after a short delay. In addition, the toxin produced a marked swelling of nodes of Ranvier of myelinated axons that reached a steady state within about 90 min, as revealed by using confocal laser scanning microscopy. The increased excitability and the nodal swelling caused by C-CTX-1 were prevented or reversed by an external hyperosmotic solution containing 100 mM D-mannitol. Moreover, the C-CTX-1-induced nodal swelling was completely prevented by the blockade of voltage-sensitive sodium channels by tetrodotoxin (TTX). It is suggested that C-CTX-1, by increasing nerve membrane excitability, enhances Na(+) entry into nodes of Ranvier through TTX-sensitive sodium channels, which directly or indirectly disturb the osmotic equilibrium between intra- and extra-axonal media resulting in an influx of water that was responsible for the long-lasting nodal swelling. The fact, that hyperosmolar D-mannitol either reversed or prevented the neurocellular actions of C-CTX-1, is of particular interest since it provides the rational basis for its use to treat the neurological symptoms of ciguatera fish poisoning in the Caribbean area.

Action Potentials↗
PubMed1999

Naftazone reduces glutamate cerebro spinal fluid levels in rats and glutamate release from mouse cerebellum synaptosomes.

It is well known that an excessive release of glutamate in the mammalian brain plays a major role in several neurological diseases. Naftazone (Etioven) is a currently used vasoprotectant drug that is metabolized in humans by reduction and glucuronidation. In the present study naftazone was found to decrease glutamate levels in the cerebro spinal fluid (CSF) of rats treated for 15 days, as determined by a chemiluminescent glutamate assay reaction. Naftazone and its glucuronide derivative also reduced respectively spontaneous and high K+-evoked glutamate release from mouse cerebellum synaptosomes. It is likely that naftazone and its glucuronide metabolite contribute in vivo to decrease glutamate levels in the CSF through their inhibitory actions on glutamate release.

Animals↗
PubMed1999

Functional repair of motor endplates after botulinum neurotoxin type A poisoning: biphasic switch of synaptic activity between nerve sprouts and their parent terminals.

Blockade of acetylcholine release by botulinum neurotoxin type A at the neuromuscular junction induces the formation of an extensive network of nerve-terminal sprouts. By repeated in vivo imaging of N-(3-triethyl ammonium propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide uptake into identified nerve endings of the mouse sternomastoid muscle after a single intramuscular injection of the toxin, inhibition of stimulated uptake of the dye at the terminals was detected within a few days, together with an increase in staining of the newly formed sprouts. After 28 days, when nerve stimulation again elicited muscle contraction, regulated vesicle recycling occurred only in the sprouts [shown to contain certain soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs) and to abut acetylcholine receptors] and not at the parent terminals. Therefore, only these sprouts could be responsible for nerve-muscle transmission at this time. However, a second, distinct phase of the rehabilitation process followed with a return of vesicle turnover to the original terminals, accompanied by an elimination of the by then superfluous sprouts. This extension and later removal of "functional" sprouts indicate their fundamental importance in the repair of paralyzed endplates, a finding with ramifications for the vital process of nerve regeneration.

Animals↗
PubMed1999

Neurotoxins targetting receptor site 5 of voltage-dependent sodium channels increase the nodal volume of myelinated axons.

The effects of a C57 type ciguatoxin (CTX-3C) and two types of brevetoxins (PbTx-1 and PbTx-3), known to bind to receptor site 5 of the neuronal voltage-dependent Na+ channel-protein, were studied on the morphology of living frog myelinated axons using confocal laser scanning microscopy. During the action of CTX-3C, PbTx-1, and PbTx-3 (10-50 nM), a marked swelling of nodes of Ranvier was observed without apparent modification of internodal parts of axons. In all cases, toxin-induced nodal swelling attained a steady-state within 75-100 min that was well maintained during an additional 90-115 min. The nodal swelling was reversed by an external hyperosmotic solution containing 100 mM D-mannitol and could be completely prevented by blocking voltage-dependent Na+ channels with 1 microM tetrodotoxin. It is suggested that CTX-3C, PbTx-1, and PbTx-3 by activating Na+ channels cause a continuous Na+ entry into axons, increasing internal Na+ concentration. Such an increase directly or indirectly disturbs the osmotic equilibrium between intra- and extra-axonal media, resulting in an influx of water, which is responsible for the long-lasting nodal swelling. Similar results were previously reported with two C60 type ciguatoxins (CTX-1B and CTX-4B). Thus, it is concluded that the four types of toxins targetting receptor site 5 of neuronal voltage-dependent Na+ channels, not only enhance nerve membrane excitability but also, on a long-term basis, cause a marked increase in the axonal volume.

Animals↗
PubMed1999

The strategy used by some piscivorous cone snails to capture their prey: the effects of their venoms on vertebrates and on isolated neuromuscular preparations.

Three piscivorous Conus species, C. ermineus, C. consor and C. catus were acclimatized in aquaria. The study of their strategy to capture the prey and details of their radula's morphology revealed that all of them used a 'hook and line' strategy which consists of immobilizing the prey rapidly before engulfing it. The venoms from these piscivorous species clearly elicit, when injected into fish, an excitotoxic shock characterized by a sudden tetanus of the prey. In mammals, the venoms induce both flaccid paralysis via i.p. injection and seizures via i.c.v. injection. Intracellular recordings from frog nerve-muscle preparations revealed that the venoms from these Conus species first caused spontaneous synaptic potentials which in turn triggered muscle action potentials. Such spontaneous activity is due to an increased nerve terminal excitability. In addition, the venoms suppressed neuromuscular transmission probably by blocking postsynaptic nicotinic acetylcholine receptors. No direct effect of these Conus venoms was observed on the membrane of skeletal muscle fibres. In conclusion, C. ermineus, C. consor and C. catus, which have not securely tethered their prey used a mixture of toxins which target both pre-and postsynaptic elements of the neuromuscular junction and which produce rapid immobilization of their prey.

Animals↗
PubMed1999

A new conotoxin isolated from Conus consors venom acting selectively on axons and motor nerve terminals through a Na+-dependent mechanism.

A novel conotoxin was isolated and characterized from the venom of the fish-hunting marine snail Conus consors. The peptide was identified by screening chromatography fractions of the crude venom that produced a marked contraction and extension of the caudal and dorsal fins in fish, and noticeable spontaneous contractions of isolated frog neuromuscular preparations. The peptide, named CcTX, had 30 amino acids and the following scaffold: X11CCX7CX2CXCX3C. At the frog neuromuscular junction, CcTx at nanomolar concentrations selectively increased nerve terminal excitability so that a single nerve stimulation triggered trains of repetitive or spontaneous synaptic potentials and action potentials. In contrast, CcTx had no noticeable effect on muscle excitability even at concentrations 100 x higher than those that affected motor nerve terminals, as revealed by direct muscle stimulation. In addition, CcTx increased miniature endplate potential (MEPP) frequency in a Ca2+-free medium supplemented with ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA). Blockade of voltage-dependent sodium channels with tetrodotoxin (TTX) either prevented or suppressed the increase of MEPP frequency induced by the toxin. CcTx also produced a TTX-sensitive depolarization of the nodal membrane in single myelinated axons giving rise, in some cases, to repetitive and/or spontaneous action potential discharges. In addition, CcTx increased the nodal volume of myelinated axons, as determined using confocal laser scanning microscopy. This increase was reversed by external hyperosmolar solutions and was prevented by pretreatment of axons with TTX. It is suggested that CcTx, by specifically activating neuronal voltage-gated sodium channels at the resting membrane potential, produced Na+ entry into nerve terminals and axons without directly affecting skeletal muscle fibres. CcTx belongs to a novel family of conotoxins that targets neuronal voltage-gated sodium channels.

Action Potentials↗
PubMed1999

[Ciguatoxins and brevetoxins: dissection of the neurobiological actions].

This review focuses on the neurobiological actions of ciguatoxins and brevetoxins which are phycotoxins produced respectively by the dinoflagellates Gambierdiscus toxicus and Ptychodiscus brevis. These actions are illustrated in particular by the effects of the toxins on myelinated nerve fibres and on skeletal neuromuscular junctions of vertebrates. Ciguatoxins and brevetoxins, through different vectors, are responsible for human intoxications characterized mainly by neurological disturbances. The molecular target of these families of lipid-soluble cyclic polyethers is the voltage-gated sodium channel, a fundamental transmembrane protein involved in cellular excitability. The different toxins share a common binding site (the receptor-site 5) located on the alpha sub-unit of this neuronal transmembrane protein. Electrophysiological studies of the mode of action of ciguatoxins and brevetoxins identify these toxins as specific sodium channel activators. Indeed, during the action of these phycotoxins, sodium channels remain permanently opened, at the resting membrane potential, which produces a continuous entry of sodium ions in most excitable cells. Such a sodium entry has various consequences on sodium-dependent physiological mechanisms, consisting in a membrane depolarization which, in turn, causes spontaneous and/or repetitive action potential discharges and thereby increases membrane excitability. These neuronal discharges may be transient or continuous according to the preparation and the toxin tested. The increase in membrane excitability during the action of ciguatoxins and brevetoxins is responsible for the different effects exerted by these toxins on various chemical synapses and secretory cells. Another consequence of the continuous entry of sodium ions into cells was revealed using confocal laser scanning microscopy and vital staining of plasma membranes with the fluorescent dye FM1-43. These techniques made feasible the dynamic study of morphological alterations produced by ciguatoxins and brevetoxins on various cellular preparations in situ. Thus, it has been possible to bring to the fore that these phycotoxins cause a marked increase in the volume of nodes of Ranvier of myelinated nerve fibres, motor nerve terminals innervating skeletal muscle and perisynaptic non-myelinating Schwann cell somata. This increase could be reversed by hyperosmotic external solutions and completely prevented by the blockade of voltage-gated sodium channels. The mechanisms involved in the increase in cellular volume, during the action of ciguatoxins and brevetoxins, are discussed.

Animals↗
PubMed1999

[Conus venoms: a source of toxins which interact with membrane- potential-dependent sodium channels].

Marine snails of the genus Conus, as they are carnivorous predators, have a venom apparatus used to capture their prey. The toxins contained in the venoms of Conidae, called conotoxins, are of a particular high degree of diversity and represent powerful tools in the neuroscience field. Indeed, these toxins specifically bind with a high affinity to receptors and ionic channels. Therefore, they provide original pharmacological tools which receive increasing investigation both to identify and study some functions of the nervous systems and to characterize new types and closely related subtypes of receptors or ionic channels. The voltage-gated sodium channel, because of its fundamental role in cell membrane excitability, is the specific target of a large number of animal and vegetal toxins. Actually, at least seven toxin receptor sites have been identified on this channel-protein. These toxins, and in particular conotoxins, are used to precise the role of different types and/or closely related subtypes of sodium channels in the peripheral and central nervous systems. The focus of the present review is to summarize our current knowledge of the consequences of physiological interactions between different conotoxin families and sodium channels.

Action Potentials↗
PubMed1999

A review on conotoxins targeting ion channels and acetylcholine receptors of the vertebrate neuromuscular junction.

In this article we present an overview of some peptides extracted and purified from the venom of marine snails of the genus Conus. These active peptides named conotoxins can be used as research tools to target voltage-gated ion channels as well as ligand-gated receptors. Because of their relatively small size, conotoxins can be chemically synthesized and made widely available. In this review we focus on conotoxins that target voltage-sensitive sodium channels, voltage-dependent calcium channels and nicotinic acetylcholine receptors of the vertebrate neuromuscular junction. Emphasis is given on summarizing our current knowledge of their primary structure and their specific pharmacological actions at the pre- and the post-synaptic level of the neuromuscular junction. Evidence is presented for conotoxins that discriminate between pre- and post-synaptic voltage-gated sodium channels. Among these peptides, the mu-conotoxin family is well characterized by its ability to block selectively sodium channels in skeletal muscle fibres without affecting axonal and nerve terminal Na+ channels. Furthermore, new conotoxins like Conus consors toxin (CcTx) and conotoxin EVIA selectively target Na+ channels in axons and nerve terminals without affecting skeletal muscle fibres. omega-conotoxins known as highly potent and selective blockers of voltage-sensitive calcium channels have proven to be valuable in determining the roles of the various subtypes of channels involved in acetylcholine release from motor nerve endings. Finally, Conus peptides which act at muscle nicotinic acetylcholine receptors constitute the most extensive characterized family of conopeptides that exhibit sequence similarity, different structural motifs and surprising diversity in their competitive and non-competitive actions.

Animals↗
PubMed1998

The wheat proteins puroindoline-a and alpha1-purothionin induce nodal swelling in myelinated axons.

The effects of two basic cysteine-rich lipid-binding proteins isolated from wheat seedlings, puroindoline-a and alpha1-purothionin, were studied on single frog myelinated axons stained with the fluorescent dye FM1-43 using confocal laser scanning microscopy. During exposure to either puroindoline-a or alpha1-purothionin (10 and 100 microM) a marked swelling of nodes of Ranvier was observed, provided NaCl was present in the external solution. It is suggested that these proteins increase the internal osmolality by forming pores in the axonal membrane and induce water influx to compensate for such an increase. Moreover, in the presence of alpha1-purothionin (100 microM), the intensity of the axonal staining with FM1-43 was increased. It is the first time, to our knowledge, that basic proteins containing domains of a cysteine-rich repeated motif are reported to produce swelling and water movements across neuronal cell membranes.

Animals↗
PubMed1998

Incorporation of synaptotagmin II to the axolemma of botulinum type-A poisoned mouse motor endings during enhanced quantal acetylcholine release.

The involvement of terminal sprouts in neurotransmitter release by in vivo botulinum type-A toxin poisoned motor endings was investigated 15 to 40 days after a single injection of the toxin onto the levator auris longus muscle of the mouse. Enhanced quantal acetylcholine release was induced by alpha-latrotoxin or La3+ in conditions that prevent endocytosis, and an antibody directed against the lumenal domain of synaptotagmin II (Syt II) was used in the presence or absence of Triton X-100. We showed that, under resting conditions, the intravesicular domain of Syt II requires Triton X-100 to be labelled, whereas it becomes exposed to the outside of the axolemma of both the original terminal arborization and the newly formed sprouts during enhanced exocytosis. These data were taken to indicate that, when sprouting is prominent, the whole modified terminal arborization, including the original branches and the sprouts, possesses the machinery for Ca2+-independent exocytosis.

Acetylcholine↗
PubMed1998

The peripheral nerve and the neuromuscular junction are affected in the tenascin-C-deficient mouse.

A thorough examination of the structure and plasticity of the neuromuscular system was performed in tenascin-C mutant mice deficient in tenascin-C. The study of the peripheral nerve revealed a number of abnormal features. In the motor nerve, numerous unmyelinated and myelinated fibers with degraded myelin were present. Schwann cell processes often enclosed degenerative terminals. Transgene (beta-galactosidase) expression analyzed at the ultrastructural level was found to be unequally distributed in the mutant's neuromuscular tissues. At the NMJ, preterminal disorganization was prevalent. Some axon terminals exhibited abnormal overgrowth. A surprising lack of beta-galactosidase expression at some cellular sites known to possess tenascin-C in wild type mice correlated best with marked changes in the cytoarchitecture of the peripheral nerve and NMJ. In some other -but not all- cellular sites which normally express the molecule, immunofluorescence analysis suggested the presence of significant but low levels of tenascin-C-like immunoreactivity together with beta-galactosidase expression. Messenger RNA detection by RT-PCR confirmed the presence of low amounts of tenascin-C mRNA in skeletal muscle suggesting that the mice deficient in tenascin-C are not complete knock-outs of this gene, but low-expression mutants. Following in vivo injections of botulinum type-A toxin, we observed a greatly reduced sprouting response of the motor nerves in tenascin-C mutant mice. We also observed that N-CAM and beta-catenin were overexpressed in the mutant. Our results suggest that tenascin-C is involved both in stabilization and in plasticity of the NMJ.

Animals↗