César Mattei

Brevenal inhibits Pacific ciguatoxin-1B-induced neurosecretion from bovine chromaffin cells

Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP) respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na+ channels through the same binding site, brevenal has therefore a good potential for the treatment of ciguatera. Pacific ciguatoxin-1B (P-CTX-1B) activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and β-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera.

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 μM 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. J. Neurosci. Res. 55:666–673, 1999. 

Xestospongin B, a competitive inhibitor of IP3-mediated Ca2+ signalling in cultured rat myotubes, isolated myonuclei, and neuroblastoma (NG108-15) cells

Xestospongin B, a macrocyclic bis‐1‐oxaquinolizidine alkaloid extracted from the marine sponge Xestospongia exigua, was highly purified and tested for its ability to block inositol 1,4,5‐trisphosphate (IP3)‐induced Ca2+ release. In a concentration‐dependent manner xestospongin B displaced [3H]IP3 from both rat cerebellar membranes and rat skeletal myotube homogenates with an EC50 of 44.6 ± 1.1 μM and 27.4 ± 1.1 μM, respectively. Xestospongin B, depending on the dose, suppressed bradykinin‐induced Ca2+ signals in neuroblastoma (NG108‐15) cells, and also selectively blocked the slow intracellular Ca2+ signal induced by membrane depolarization with high external K+ (47 mM) in rat skeletal myotubes. This slow Ca2+ signal is unrelated to muscle contraction, and involves IP3 receptors. In highly purified isolated nuclei from rat skeletal myotubes, Xestospongin B reduced, or suppressed IP3‐induced Ca2+ oscillations with an EC50 = 18.9 ± 1.35 μM. In rat myotubes exposed to a Ca2+‐free medium, Xestospongin B neither depleted sarcoplasmic reticulum Ca2+ stores, nor modified thapsigargin action and did not affect capacitative Ca2+ entry after thapsigargin‐induced depletion of Ca2+ stores. Ca2+‐ATPase activity measured in skeletal myotube homogenates remained unaffected by Xestospongin B. It is concluded that xestospongin B is an effective cell‐permeant, competitive inhibitor of IP3 receptors in cultured rat myotubes, isolated myonuclei, and neuroblastoma (NG108‐15) cells.

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 × 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‐(β‐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.

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.

Gambiertoxin (CTX-4B), purified from wild Gambierdiscus toxicus dinoflagellates, induces Na(+)-dependent swelling of single frog myelinated axons and motor nerve terminals in situ.

The effects of gambiertoxin (CTX-4B), purified from the dinoflagellate Gambierdiscus toxicus, were assessed on the morphology of both frog myelinated axons and motor nerve terminals, using confocal laser scanning microscopy. During the action of the toxin (24 and 30 nM), a marked swelling of nodes of Ranvier and motor nerve terminals was observed. The CTX-4B-induced swelling could be prevented by blocking voltage-dependent Na+ channels with tetrodotoxin, and could be partly reversed by an external hyperosmotic solution containing 100 mM D-mannitol. The results suggest that CTX-4B, by modifying voltage-dependent Na+ channels, increases internal Na+ concentration of axons and nerve terminals and consequently induces water influx to compensate such an increase. It is suggested that stimulated transmitter release by CTX-4B, as well as by hyperosmotic dmannitol, contribute also to the swelling of the terminals through an increase in their surface area.

Dual action of a dinoflagellate-derived precursor of Pacific ciguatoxins (P-CTX-4B) on voltage-dependent K+ and Na+ channels of single myelinated axons

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.

The wheat proteins puroindoline-a and α1-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.

Ciguatoxin-induced catecholamine secretion in bovine chromaffin cells: mechanism of action and reversible inhibition by brevenal

Ciguatoxin (P-CTX-1B) from the dinoflagellate Gambierdiscus toxicus, belongs to the family of polyether neurotoxins responsible for the neurological poisoning disorder ciguatera. Although it is the most widespread marine-borne disease affecting humans, there is no current FDA-approved treatment available except for symptomatic therapies. In this paper, we report that P-CTX-1B promotes catecholamine secretion from bovine chromaffin cells, an effect that is insensitive to concomitant activation of capacitative Ca(2+) entry. Moreover, we confirm that brevenal, a polyether from the dinoflagellate Karenia brevis, blocks P-CTX-1B-induced catecholamine secretion. This effect is partially reversible. Our results therefore raise the prospect of finding functional antagonists for P-CTX-1B that could be useful for the treatment of ciguatera.

Analysis of Caribbean ciguatoxin-1 effects on frog myelinated axons and the neuromuscular junction.

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.