Neuromuscular blocking action of the Theraphosa blondii spider venom

In 1919, Houssay and Negrete reported that venoms of Theraphosidae spiders induced neuromuscular blockade. In 1993, a purified toxin from Grammostola spider venom was found to block the P-type voltage-dependent calcium channel (VDCC), causing neuromuscular blockade. We studied the mode of action of Theraphosa blondii venom, a large Theraphosidae spider from Northern Brazil, Venezuela, and The Guyanas in mouse phrenic nerve-diaphragm preparation. This venom elicited a partially reversible neuromuscular blockade and did not depress directly evoked twitches or alter the membrane potential. Neostigmine produced only a poor antagonistic effect on partially blocked diaphragms. However, completely blocked miniature endplate potentials (m.e.p.ps) were reverted by neostigmine. These results can be explained by the presence of toxins in the venom that interact with the endplate receptor at the acetylcholine sites (curareminetic toxins) and toxins that inhibit the P-type voltage-dependent calcium channel (VDCC) (ômega-toxins). This study shows that Theraphosidae venoms, especially those of the Theraphosa blondii, are a source of curaremimetic toxins and ômega-toxins of possible interest as tools in bioscientific research.

Nature of the postsynaptic action of crotoxin at guinea-pig diaphragm end-plates.

Crotoxin is known to desensitize the nicotinic receptor of Torpedo marmorata and Electrophorus electricus electroplaques. The aim of the present study was to elucidate whether the postsynaptic effect of crotoxin at a mammalian muscle end-plate is also caused by receptor desensitization or results from a curaremimetic action. For this purpose, we investigated the action of 4-aminopyridine (4-AP) on crotoxin-induced blockade of miniature end-plate potentials (m.e.p.p.s) and of the depolarization of end-plates produced by carbachol. The experiments were carried out in guinea-pig diaphragms bathed in Tyrode solution at 37 degrees C and gassed with 95% O2, 5% CO2. The potentials were measured with conventional techniques using glass microelectrodes. Even at low concentrations, crotoxin blocked the m.e.p.p.s and this blockade was antagonized by 4-AP. Neostigmine was without effect. 4-AP did not restore the m.e.p.p.s blocked by either d-tubocurarine (dTc) or beta-bungarotoxin (beta-BTX). 4-AP also antagonized the crotoxin-induced blockade of the end-plate depolarization produced by carbachol. These results show that the postsynaptic effect of crotoxin at the guinea-pig muscle end-plate also results from nicotinic receptor desensitization.

The neuromuscular action of Ancylometes sp. spider venom in the rat phrenic nerve-diaphragm preparation.

The effects of Ancylometes sp. venom on muscle contraction and bioelectrical potentials were investigated in the rat phrenic nerve diaphragm muscle preparation. The venom (50 microg/ml) depolarized the diaphragm muscle fiber membranes. This effect was abolished by tetrodotoxin and by reduction of the sodium concentration of the Tyrode solution. The increase in the frequency of miniature end plate potentials induced by the venom was also suppressed by tetrodotoxin (3 microM). These results indicate that the venom may activate voltage-dependent sodium channels in cell membranes. All of the effects of Ancylometes sp. venom on this nerve muscle preparation (i.e. increase in twitch tension, spontaneous small phasic contractions and increase in the frequency of miniature end-plate potentials) may be explained in terms of its action on sodium channels.

Pathological changes induced by PhTx1 from Phoneutria nigriventer spider venom in mouse skeletal muscle in vitro.

The 'armed' spider Phoneutria nigriventer is responsible for most human accidents involving spiders in Brazil. The effects of fraction Tx1 (PhTx1) from the venom of this spider were investigated by physiological and morphological methods using the mouse phrenic nerve-diaphragm preparation. PhTx1 (1 and 5 microg) did not affect the twitch tension of muscle fibers under indirect electrical stimulation. At this same concentration, PhTx1 also did not alter the miniature end-plate potential (mepp) frequency and amplitude, nor did it change the resting membrane potential 60 min after addition to the preparation. Light microscopy (LM) revealed that in muscles incubated with PhTx1 a number of fibers were morphologically altered, as evidenced by microvacuolization and myofibril hypercontraction and loss within 15 min after toxin administration. Transmission electron microscopy (TEM) showed sarcoplasmic reticulum swelling, disorganization of the sarcomeres and mitochondrial damage, and occasionally, sarcolemmal discontinuities with a persisting basal membrane. The intra-muscular fascicles of the phrenic nerve showed myelinated axons with vacuolated myelin sheaths as well as peri- and intra-axoplasmic vacuoles. The neuromuscular junction changes were variable, but were rarely severe. Thus, although PhTx1 did not depolarize or hyperpolarize the neuromuscular junction, it was nevertheless toxic to a restricted number of muscle fibers and nerve structures. The site of action of PhTx1 may involve the sarcolemma and axolemma as suggested by the morphological abnormalities which could reflect hydroelectrolytic disturbances.

Mode of action of Duvernoy's gland extracts from the colubrid Dryadophis bifossatus in the chick biventer cervicis nerve-muscle preparation.

Extracts and secretion of the Duvernoy's gland are venomous, inducing in several cases motor paralysis in experimental animals. The extracts of the Duvernoy's gland from the aglyphous colubrid Dryadophis bifossatus are very toxic, eliciting flaccid paralysis in pigeons, rabbits and Hylae (Brazil and Vellard, 1926). In the present study, the neuromuscular action of the extracts of Duvernoy's gland from this Colubridae was investigated in the chick biventer cervicis nerve-muscle preparation. The muscle was indirectly stimulated with supramaximal pulses, and by addition of acetylcholine or carbachol to the organ bath. Direct muscle stimulation was carried out in curarized preparations. The extracts induced an irreversible neuromuscular blockade and also inhibited irreversibly the contracture of the biventer cervicis produced by either acetylcholine or carbachol. The twitches elicited by direct muscle stimulation were not depressed. These results show that the neurotoxin(s) of the extracts interact with the end-plate cholinergic receptors.

Toxins as tools in the study of sodium channel distribution in the muscle fibre membrane.

The number of tetrodotoxin molecules bound to the membrane of the fibres of muscles in normal conditions and after detubulation produced by glycerol-induced osmotic shock pointed to a higher sodium channel density at the surface membrane than at the membrane in the transverse tubules. Study of the maximum rate of rise of the action potential at the junctional and nonjunctional regions of the muscle fibre membrane suggested that the Na+ channel density is also not the same along the muscle fibre membrane, being higher at the junctional region. Further studies on the distribution of the Na+ channel along the muscle fibre membrane were carried out with the use of (1) the loose patch voltage-clamp technique, (2) labelling the Na+ channels with fluorescently labelled scorpion toxins, (3) autoradiography of localized Na+ channels with 125I-labelled scorpion toxins, and (4) toxins that induce persistent activation of the Na+ channel. The studies referred to in (1), (2) and (3) demonstrate that the density of the Na+ channel is much higher at the junctional region than elsewhere in the membrane of the muscle fibre. On the other hand, in experiments carried out on curarized rat diaphragms several sodium channel activating toxins (crotamine, Phoneutria nigriventer venom, its toxin PhTx2, veratrine) were found to produce a much greater depolarization of the membrane at the junctional region than at nonjunctional regions. However, it was also found that some toxins (veratridine, batrachotoxin) depolarized equally well the junctional and nonjunctional regions. Two alternative hypotheses to explain the uniform depolarization of the muscle fibre membrane induced by these toxins are suggested.

Effects of Phoneutria nigriventer venom on rabbit vascular smooth muscle.

1. The effects of Phneutria nigriventer venom (PNV) on rabbit vascular smooth muscle have been investigated. De-endothelialized vascular strips were superfused in a cascade system with oxygenated (95% O2 + 5% CO2) Krebs solution at 37 degrees C. 2. Phoneutria nigriventer venom (0.3-30 micrograms) produced dose-dependent and short-lived contractions of both venous (cava, mesenteric and jugular veins) and arterial (pulmonary and mesenteric arteries) tissues. 3. Methysergide (5.0 microM) did not significantly affect PNV-induced contractions in venous tissues (cava and mesenteric veins) or pulmonary artery, indicating that serotonin is not involved in the contraction. This was confirmed when PNV was dialyzed (24-48 h) since the contracting activity was still observed on the above tissues. In addition, the spasmogenic activity induced by dialyzed PNV was greatly reduced by incubating the venom with trypsin. 4. Neither tetrodotoxin (3.0 microM) nor phenoxybenzamine (0.05 microM) significantly affected PNV-induced contractions, suggesting that voltage-dependent sodium channel activation or endogenous catecholamine release from autonomic nerve endings on the vascular walls do not play a role in the response to PNV. 5. Our results demonstrate that PNV contains non-dialyzable components, probably peptides, that are responsible for the contractile activity on rabbit veins and pulmonary artery strips.

Effects of Phoneutria nigriventer venom on rabbit vascular smooth muscle

1. The effects of Phneutria nigriventer venom (PNV) on rabbit vascular smooth muscle have been investigated. De-endothelialized vascular strips were superfused in a cascade system with oxygenated (95 per cent O2 + 5 per cent CO2) Krebs solution at 37 degrees C. 2. Phoneutria nigriventer venom (0.3-30 micrograms) produced dose-dependent and short-lived contractions of both venous (cava, mesenteric and jugular veins) and arterial (pulmonary and mesenteric arteries) tissues. 3. Methysergide (5.0 microM) did not significantly affect PNV-induced contractions in venous tissues (cava and mesenteric veins) or pulmonary artery, indicating that serotonin is not involved in the contraction. This was confirmed when PNV was dialyzed (24-48 h) since the contracting activity was still observed on the above tissues. In addition, the spasmogenic activity induced by dialyzed PNV was greatly reduced by incubating the venom with trypsin. 4. Neither tetrodotoxin (3.0 microM) nor phenoxybenzamine (0.05 microM) significantly affected PNV-induced contractions, suggesting that voltage-dependent sodium channel activation or endogenous catecholamine release from autonomic nerve endings on the vascular walls do not play a role in the response to PNV. 5. Our results demonstrate that PNV contains non-dialyzable components, probably peptides, that are responsible for the contractile activity on rabbit veins and pulmonary artery strips

Effect of 4-aminopyridine on the postsynaptic action of polymyxin B.

Polymyxin B produces neuromuscular blockade by acting at pre- and postsynaptic sites. Its postsynaptic effect has been attributed either to blockade of open ionic channels or to the conversion of the end-plate receptor from a low to a high affinity state, i.e. to a state similar to the desensitized one caused by agonists. Since 4-aminopyridine inhibits agonist-induced end-plate receptor desensitization, we decided to investigate its effect on the postsynaptic action of polymyxin B. The experiments were carried out on the isolated rat diaphragm; miniature end-plate potentials (m.e.p.p.s) and carbachol-induced depolarization at the end-plate region were recorded with microelectrode techniques. Polymyxin B, 40 micrograms/ml reduced the amplitude of the m.e.p.p.s and suppressed them in about 30 min. 4-Aminopyridine completely antagonized this effect. Neostigmine did not restore the m.e.p.p.s suppressed by polymyxin B nor did 4-aminopyridine antagonize the effect of d-tubocurarine on these potentials. The carbachol-induced depolarization was blocked only partially by polymyxin B. 4-Aminopyridine B nearly completely antagonized this effect. The antagonistic effect of 4-aminopyridine on the postsynaptic action of polymyxin B favors the receptor desensitization' hypothesis.

Effects of the venom of the Brazilian scorpion Tityus serrulatus and two of its fractions on the isolated diaphragm of the rat.

1. The effects of Tityus serrulatus venom and of two of its toxic fractions, toxin gamma (Tx gamma) and T2III1, on the rat isolated diaphragm were examined. 2. The crude venom (5 ng) facilitated the neuromuscular transmission and increased the twitch tension evoked by retrograde injection of Ach. 3. Tx gamma (25-100 ng) and fraction T2III1 (2.5 ng) also facilitated the neuromuscular transmission but only fraction T2III1 increased the twitch tension evoked by retrograde injection of Ach. 4. Tx gamma (50 ng) and fraction T2III1 (2.5 ng) produced a tetrodotoxin-sensitive increase in the frequency of miniature endplate potentials (m.e.p.p.) and a transitory reduction of the resting potential. The latter effect of the fractions was prevented by treating muscles with tetrodotoxin or D-tubocurarine. Fraction T2III1 also produced a tetrodotoxin-resistance increase of m.e.p.p. amplitude. 5. These results suggest that Tx gamma enhances Ach output through the activation of Na+ channels in the motor nerve terminals. Fraction T2III1 produced effects similar to those induced by Tx gamma but also acted at postjunctional sites, probably by increasing subsynaptic membrane sensitivity to the neurotransmitter.

Unequal depolarization of the membrane of the rat diaphragm muscle fibres caused by veratrine.

The effect produced by veratrine on transmembrane potential was investigated in five distinct regions of the isolated rat diaphragm blocked with either d-tubocurarine or alpha-bungarotoxin. It was found that small (0.4 micrograms/ml) and large (2.0 micrograms/ml) doses of veratrine depolarize only two or three of these regions. With the use of a very large (10.0 micrograms/ml) dose depolarization occurred in all five regions of the diaphragm but the effect was much larger in those areas of the muscle fibre membrane which were selectively depolarized by the smaller ones. These results clearly indicate either an unequal distribution of sodium channels activated by veratrine or differences in sodium channel density in distinct areas of the muscle fibre membrane. Supersensitivity of veratrinized muscles to potassium was confirmed. However, the sensitiveness to the depolarizing action of potassium was only increased in the regions of the diaphragm partially depolarized by veratrine. This suggests that partial depolarization of the muscle fibre membrane was actually the cause of the supersensitivity. The possible involvement of potassium and of the unequal depolarization of the muscle fibre membrane in the veratrine response is considered.

Mode of action of Phoneutria nigriventer spider venom at the isolated phrenic nerve-diaphragm of the rat.

The effects of Phoneutria nigriventer venom on muscle contraction and bioelectrical potentials were investigated in the rat phrenic nerve-diaphragm muscle preparation. The venom caused a non-uniform depolarization of the diaphragm muscle fiber membrane. This effect was abolished by tetrodotoxin or reduction of the sodium concentration in the bath fluid. The increase in the frequency of miniature end-plate potentials induced by the venom was also suppressed by tetrodotoxin. These results indicate that the venom activates the voltage-dependent sodium channel in muscle and nerve cell membranes. All the effects of the venom on the phrenic nerve-diaphragm muscle preparation (i.e. increase in twitch tension, delay in twitch relaxation, initial tonic contraction of short duration, spontaneous small phasic contractions, blockade of neuromuscular transmission, repetitive firing in nerve and muscle fiber membranes) can be explained on the basis of its action in the sodium channel. Nearly all of these effects are caused by discharges of repetitive action potentials in the nerve and/or muscle fiber membranes.

Effect of 4-aminopyridine on end-plate receptor desensitization caused by carbachol.

The effect of 4-aminopyridine on receptor desensitization was investigated in the isolated rat diaphragm by measuring transmembrane potential in the end-plate region. When 4-aminopyridine was added to the bath before carbachol, the depolarization produced by the agonist was permanent, that is 4-aminopyridine completely inhibited the receptor desensitization produced by prolonged exposure of the receptors to carbachol. When it was added after carbachol in the phase in which the membrane had already repolarized, the end-plate region depolarized. Therefore, 4-aminopyridine was also able to reverse the receptors from the desensitized state to the resting non-desensitized one. The effect of 4-aminopyridine was concentration-dependent. Calcium antagonized the effect of 4-aminopyridine. 4-Aminopyridine also reversed the receptors from their desensitized state to the resting one in the isolated and chronically denervated rat hemidiaphragm. The known action of the aminopyridines cannot explain the effect of 4-aminopyridine described here. It is suggested that it is caused by an action on either the receptor-ionic channel complex or the lipids of the postjunctional membrane surrounding it.