Mouse Phrenic Nerve-hemidiaphragm Preparations Research Articles

Comparative electrophysiological characterization of ammodytoxin A, a β-neurotoxin from the nose-horned viper venom, and its enzymatically inactive mutant

Presynaptic- or β-neurotoxicity of secreted phospholipases A2 (sPLA2) is a complex process. For full expression of β-neurotoxicity, the enzymatic activity of the toxin is essential. However, it has been shown that not all toxic effects of a β-neurotoxin depend on its enzymatic activity, for example, the inhibition of mitochondrial cytochrome c oxidase. The main objective of this study was to verify whether it is possible to observe and study the phospholipase-independent actions of β-neurotoxins by a standard ex vivo twitch-tension experimental approach. To this end, we compared the effects of a potent snake venom β-neurotoxin, ammodytoxin A (AtxA), and its enzymatically inactive mutant AtxA(D49S) on muscle contraction of the mouse phrenic nerve-hemidiaphragm preparation. While AtxA significantly affected the amplitude of the indirectly evoked isometric muscle contraction, the resting tension of the neuromuscular (NM) preparation, the amplitude of the end-plate potential (EPP), the EPP half decay time and the resting membrane potential, AtxA(D49S) without enzymatic activity did not. From this, we can conclude that the effects of AtxA independent of enzymatic activity cannot be studied with classical electrophysiological measurements on the isolated NM preparation. Our results also suggest that the inhibition of cytochrome c oxidase activity by AtxA is not involved in the rapid NM blockade by this β-neurotoxin, but that its pathological consequences are rather long-term. Interestingly, in our experimental setup, AtxA upon direct stimulation reduced the amplitude of muscle contraction and induced contracture of the hemidiaphragm, effects that could be interpreted as myotoxic.

The Safety Factor for Neuromuscular Transmission: Effects of Dimethylsulphoxide, Cannabinoids and Synaptic Homeostasis.

BackgroundIn myasthenia gravis, impaired postsynaptic sensitivity to acetylcholine results in failure of neuromuscular transmission and fatiguing muscle weakness.ObjectiveDevelop an ex vivo muscle contraction assay to test cannabinoids and other substances that might act on the myasthenic neuromuscular junction to restore control of the muscle.MethodsTubocurarine was added to an ex vivo, mouse phrenic nerve-hemidiaphragm muscle preparation to reduce acetylcholine sensitivity. This produced a myasthenia-like decrement in twitch force during a train of 10 nerve impulses (3 / sec). Endplate potential (EPP) recordings were used to confirm and extend the findings.ResultsSurprisingly, addition to the bath of dimethylsulphoxide (DMSO), at concentrations as low as 0.1%(v/v), partially reversed the decrement in nerve-evoked force. Intracellular electrophysiology, conducted in the presence of tubocurarine, showed that DMSO increased the amplitudes of both the spontaneous miniature EPP (MEPP) and the (nerve-evoked) EPP. In the absence of tubocurarine (synaptic potentials at physiological levels), an adaptive fall in quantal content negated the DMSO-induced rise in EPP amplitude. The effects of cannabinoid receptor agonists (solubilized with DMSO) in the contraction assay do not support their further exploration as useful therapeutic agents for myasthenia gravis. CP 55,940 (a dual agonist for cannabinoid receptor types 1 and 2) reversed the beneficial effects of DMSO.Conclusions:We demonstrate a powerful effect of DMSO upon quantal amplitude that might mislead pharmacological studies of synaptic function wherever DMSO is used as a drug vehicle. Our results also show that compounds targeting impaired neuromuscular transmission should be tested under myasthenic-like conditions, so as to avoid confounding effects of synaptic homeostasis.

Botulinum neurotoxin serotype D – A potential treatment alternative for BoNT/A and B non-responding patients

ObjectivesBotulinum neurotoxin serotypes A and B (BoNT/A & B) are highly effective medicines to treat hyperactive cholinergic neurons. Due to neutralizing antibody formation, some patients may become non-responders. In these cases, the serotypes BoNT/C-G might become treatment alternatives. BoNT/D is genetically least related to BoNT/A & B and thereby circumventing neutralisation in A/B non-responders. We produced BoNT/D and compared its pharmacology with BoNT/A ex vivo in mice tissue and in vivo in human volunteers. MethodsBoNT/D was expressed recombinantly in E. coli, isolated by chromatography and its ex vivo potency was determined at mouse phrenic nerve hemidiaphragm preparations. Different doses of BoNT/D or incobotulinumtoxinA were injected into the extensor digitorum brevis (EDB) muscles (n = 30) of human volunteers. Their compound muscle action potentials were measured 11 times by electroneurography within 220 days. ResultsDespite a 3.7-fold lower ex vivo potency in mice, a 110-fold higher dosage of BoNT/D achieved the same clinical effect as incobotulinumtoxinA while showing a 50% shortened duration of action. ConclusionsBoNT/D blocks dose-dependently acetylcholine release in human motoneurons upon intramuscular administration, but its potency and duration of action is inferior to approved BoNT/A based drugs. SignificanceBoNT/D constitutes a potential treatment alternative for BoNT/A & B non-responders.

In vitro and ex vivo screening of candidate therapeutics to restore neurotransmission in nerve terminals intoxicated by botulinum neurotoxin serotype A1

Botulinum neurotoxins (BoNTs) are exceedingly potent neurological poisons that block cholinergic release in the peripheral nervous system and cause death by asphyxiation. While post-exposure prophylaxis can effectively eliminate toxin in the bloodstream, there are no clinically effective treatments to prevent or reverse disease once BoNT has entered the neuron. To address the need for post-symptomatic countermeasures, we designed and developed an in vitro assay based on whole-cell, patch-clamp electrophysiological monitoring of miniature excitatory post-synaptic currents in synaptically active murine embryonic stem cell-derived neurons. This synaptic function-based assay was used to assess the efficacy of rationally selected drugs to restore neurotransmission in neurons comprehensively intoxicated by BoNT/A. Based on clinical reports suggesting that elevated Ca2+ signaling promotes symptomatic relief from botulism, we identified seven candidate drugs that modulate presynaptic Ca2+ signaling and assessed their ability to reverse BoNT/A-induced synaptic blockade. The most effective drugs from the screen were found to phasically agonize voltage-gated calcium channel (VGCC) activity. Lead candidates were then applied to ex vivo studies in BoNT/A-paralyzing mouse phrenic nerve-hemidiaphragm (PND) preparations. Treatment of PNDs with VGCC agonists after paralytic onset transiently potentiated nerve-elicited muscle contraction and delayed progression to neuromuscular failure. Collectively, this study suggests that Ca2+-modulating drugs represent a novel symptomatic treatment for neuromuscular paralysis following BoNT/A poisoning.

Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library

Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease.

Identification of the SV2 protein receptor-binding site of botulinum neurotoxin type E

The highly specific binding and uptake of BoNTs (botulinum neurotoxins; A-G) into peripheral cholinergic motoneurons turns them into the most poisonous substances known. Interaction with gangliosides accumulates the neurotoxins on the plasma membrane and binding to a synaptic vesicle membrane protein leads to neurotoxin endocytosis. SV2 (synaptic vesicle glycoprotein 2) mediates the uptake of BoNT/A and /E, whereas Syt (synaptotagmin) is responsible for the endocytosis of BoNT/B and /G. The Syt-binding site of the former was identified by co-crystallization and mutational analyses. In the present study we report the identification of the SV2-binding interface of BoNT/E. Mutations interfering with SV2 binding were located at a site that corresponds to the Syt-binding site of BoNT/B and at an extended surface area located on the back of the conserved ganglioside-binding site, comprising the N- and C-terminal half of the BoNT/E-binding domain. Mutations impairing the affinity also reduced the neurotoxicity of full-length BoNT/E at mouse phrenic nerve hemidiaphragm preparations demonstrating the crucial role of the identified binding interface. Furthermore, we show that a monoclonal antibody neutralizes BoNT/E activity because it directly interferes with the BoNT/E-SV2 interaction. The results of the present study suggest a novel mode of binding for BoNTs that exploit SV2 as a cell surface receptor.

Effects of odontobuthus doriae scorpion venom on mouse sciatic nerve.

Temporary paralysis is a rare manifestation of envenoming following the yellow Iranian scorpion, Odontobuthus doriae (O. doriae). Thus, to elucidate the underlying mechanism, we investigated the neurotoxic effect of venom in the sciatic nerve, the possible mechanism in a mice model. The neurotoxicity and temperature effects in the venom-induced neurotoxicity were examined using the mouse sciatic nerve and mouse phrenic nerve-hemidiaphragm (MHD) preparations. O .doriae venom (1 μg/mL) caused changes in the perineural waveform associated with nerve terminal action potentials. Venom affected on both negative and positive components of the waveform which is known as a compound action potential. The timeresponse relationship of venom-induced depression of resting membrane potential (RMP) was significant (p < 0.05). No significant difference in augmentation was seen in room temperature in comparison with 37°C. In conclusion, although there was no evidence that the venom had any specific curarizing action at the neuromuscular junction, the results suggest that the venom exerts its neuromuscular transmission on the sciatic nerve through potassium and sodium ionic-currents. Furthermore, the influence of temperature on neurotoxicity was ineffective on blockade of the neuromuscular transmission in-vitro.

Reversal of BoNT/A-mediated inhibition of muscle paralysis by 3,4-diaminopyridine and roscovitine in mouse phrenic nerve-hemidiaphragm preparations

Botulinum neurotoxins (BoNTs) comprise a family of neurotoxic proteins synthesized by anaerobic bacteria of the genus Clostridium. Each neurotoxin consists of two polypeptide chains: a 100kDa heavy chain, responsible for binding and internalization into the nerve terminal of cholinergic motoneurons and a 50kDa light chain that mediates cleavage of specific synaptic proteins in the host nerve terminal. Exposure to BoNT leads to cessation of voltage- and Ca2+-dependent acetylcholine (ACh) release, resulting in flaccid paralysis which may be protracted and potentially fatal.There are no approved therapies for BoNT intoxication once symptoms appear, and specific inhibitors of the light chain developed to date have not been able to reverse the consequences of BoNT intoxication. An alternative approach for treatment of botulism is to focus on compounds that act by enhancing ACh release. To this end, we examined the action of the K+ channel blocker 3,4-diaminopyridine (3,4-DAP) in isolated mouse hemidiaphragm muscles intoxicated with 5pM BoNT/A. 3,4-DAP restored tension within 1–3min of application, and was effective even in totally paralyzed muscle. The Ca2+ channel activator (R)-roscovitine (Ros) potentiated the action of 3,4-DAP, allowing for use of lower concentrations of the K+ channel blocker. In the absence of 3,4-DAP, Ros was unable to augment tension in BoNT/A-intoxicated muscle. This is the first report demonstrating the efficacy of the combination of 3,4-DAP and Ros for the potential treatment of BoNT/A-mediated muscle paralysis.

Venom Properties of the Spider &lt;I&gt;Latrodectus tredecimguttatus&lt;/I&gt; and Comparison of Two Venom-Collecting Methods

The bioactivities and proteinous compositions of venoms collected by two different methods from Latrodectus tredecimguttatus were analyzed and compared. Most of proteinous venom components were high-molecular-mass acidic proteins (>10⁴). Compared with the venom obtained from dissected venom gland (DGV), the venom obtained by electrical stimulation (ESV) contained more high molecular mass proteins, but the venom proteins and peptides with low molecular mass (<10⁴) in the preparations were very similar. Intraperitoneal injection of the two venoms in mice gave rise to similar poisoning symptoms and the LD₅₀ values of ESV and DGV were (0.16&plusmn;0.03) mg/kg and (0.39&plusmn;0.05) mg/kg, respectively. LD₅₀ values of ESV and DGV in cockroaches (Periplaneta Americana) were 1.87 &mu;g/g and 2.32 &mu;g/g, respectively. ESV was able to block neuromuscular transmission in the mouse phrenic nerve-hemidiaphragm preparation within (25.0&plusmn;2.2) min at a concentration of 3.2&times;10^-6g/mL, whereas DGV blocked neuromuscular transmission within (45.7&plusmn;1.8 )min at the same concentration. The fraction of the venom containing proteins and peptides with molecular mass <10⁴ did not obviously affect the transmission. Those results demonstrated that Latrodectus tredecimguttatus venom was a mixture rich in larger proteins rather than smaller proteins and peptides; the mammalian toxicities of the venom were due primarily to the high molecular weight acidic proteins rather than low molecular weight peptides; the active components contained in ESV and DGV were similar but the content in ESV was higher than that in DGV.

Evaluation of toosendanin as a botulinum neurotoxin antagonist

Toosendanin (TSN), a tetranortriterpenoid compound from the plant Melia toosendan, has been reported to protect rats and non-human primates from the lethal actions of botulinum neurotoxin A (BoNT/A). TSN was tested in isolated mouse phrenic nerve-hemidiaphragm preparations against BoNT serotypes A, B and E. When added prior to toxin, TSN was found to delay the onset of muscle paralysis with all 3 BoNT serotypes. TSN had little or no effect on BoNT-induced muscle paralysis when applied 30 min after BoNT. It is suggested that the antagonist activity of TSN may result from inhibition of BoNT translocation.

Ability of rabbit antiserum against crotapotin to neutralize the neurotoxic, myotoxic and phospholipase A 2 activities of crotoxin from Crotalus durissus cascavella snake venom

The toxicity of crotoxin, the major toxin of Crotalus durissus terrificus (South American rattlesnake) venom, is mediated by its basic phospholipase A 2 (PLA 2) subunit. This PLA 2 is non-covalently associated with crotapotin, an acidic, enzymatically inactive subunit of the crotoxin complex. In this work, rabbit antiserum raised against crotapotin purified from Crotalus durissus cascavella venom was tested for its ability to neutralize the neurotoxicity of this venom and its crotoxin in vitro. The ability of this antiserum to inhibit the enzymatic activity of the crotoxin complex and PLA 2 alone was also assessed, and its potency in preventing myotoxicity was compared with that of antisera raised against crotoxin and PLA 2. Antiserum to crotapotin partially neutralized the neuromuscular blockade caused by venom and crotoxin in electrically stimulated mouse phrenic nerve-hemidiaphragm preparations and prevented the venom-induced myotoxicity, but did not inhibit the enzymatic activity of crotoxin and purified PLA 2. In contrast, previous findings showed that antisera against crotoxin and PLA 2 from C. d. cascavella effectively neutralized the neuromuscular blockade and PLA 2 activity of this venom and its crotoxin. The partial neutralization of crotoxin-mediated neurotoxicity by antiserum to crotapotin probably reduced the binding of crotoxin to its receptor following interaction of the antiserum with the crotapotin moiety of the complex.

Binding to the high-affinity M-type receptor for secreted phospholipases A 2 is not obligatory for the presynaptic neurotoxicity of ammodytoxin A

R180, isolated from porcine brain cortex, is a high-affinity membrane receptor for ammodytoxin A (AtxA), a secreted phospholipase A 2 (sPLA 2) and presynaptically active neurotoxin from venom of the long-nosed viper ( Vipera ammodytes ammodytes). As a member of the M-type sPLA 2 receptors, present on the mammalian plasma membrane, R180 has been proposed to be responsible for one of the first events in the process of presynaptic neurotoxicity, the binding of the toxin to the nerve cell. To test this hypothesis, we prepared and analyzed three N-terminal fusion proteins of AtxA possessing a 12 or 5 amino acid residue peptide. The presence of such an additional “propeptide” prevented interaction of the toxin with the M-type receptor but not its lethality in mouse and neurotoxic effects on a mouse phrenic nerve-hemidiaphragm preparation. In addition, antibodies raised against the sPLA 2-binding C-type lectin-like domain 5 of the M-type sPLA 2 receptor were unable to abolish the neurotoxic action of AtxA on the neuromuscular preparation. The specific enymatic activities of the fusion AtxAs were two to three orders of magnitude lower from that of the wild type, yet resulting in a similar but less pronounced neurotoxic profile on the neuromuscular junction. This is in accordance with other data showing that a minimal enzymatic activity suffices for presynaptic toxicity of sPLA 2s to occur. Our results indicate that the interaction of AtxA with the M-type sPLA 2 receptor at the plasma membrane is not essential for presynaptic activity of the toxin. Interaction of AtxA with two intracellular proteins, calmodulin and the R25 receptor, was affected but not prevented by the presence of the N-terminal fusion peptides, implying that these proteins may play a role in the sPLA 2 neurotoxicity.

Adenosine decreases both presynaptic calcium currents and neurotransmitter release at the mouse neuromuscular junction.

A controversy currently exists as to the mechanism of action by which adenosine, an endogenous mediator of neurotransmitter depression, reduces the evoked release of the neurotransmitter acetylcholine (ACh) at the skeletal neuromuscular junction. Specifically, it is uncertain whether adenosine inhibits ACh release from mammalian motor nerve endings by reducing Ca(2+) calcium entry through voltage-gated calcium channels or, as is the case at amphibian motor nerve endings, by an effect downstream of Ca(2+) entry. In an attempt to address this controversy, the effects of adenosine on membrane ionic currents and neurotransmitter release were studied at neuromuscular junctions in adult mouse phrenic nerve hemidiaphragm preparations. In wild-type mice, adenosine (500 microm-1 mm) reduced prejunctional Ca(2+) currents simultaneously with a reduction in evoked ACh release. In Rab3A knockout mice, which have been shown to have an increased sensitivity to adenosine, the simultaneous reduction in Ca(2+) currents and ACh secretion occurred at significantly lower adenosine concentrations (< or = 50 microM). Measurements of nerve terminal Na(+) and K(+) currents made simultaneously with evoked ACh release demonstrated that the decreases in Ca(2+) currents were not attributable to changes in cation entry through voltage-gated Na(+) or K(+) channels. Furthermore, no effects of adenosine on residual ionic currents were observed when P/Q-type calcium channels were blocked by Cd(2+) or omega-agatoxin-IVA. The results demonstrate that inhibition of evoked neurotransmitter secretion by adenosine is associated with a reduction in Ca(2+) calcium entry through voltage-gated P/Q Ca(2+) channels at the mouse neuromuscular junction. Whilst it may be that adenosine inhibits ACh release by different mechanisms at amphibia and mammalian neuromuscular junctions, it is also possible that the secretory apparatus is more intimately coupled to the Ca(2+) channels in the mouse such that an effect on the secretory machinery is reflected as changes in Ca(2+) currents.

Functional characterisation of tetanus and botulinum neurotoxins binding domains.

Tetanus and botulinum neurotoxins constitute a family of bacterial protein toxins responsible for two deadly syndromes in humans (tetanus and botulism, respectively). They bind with high affinity to neurons wherein they cause a complete inhibition of evoked neurotransmitter release. Here we report on the cloning, expression and use of the recombinant fragments of the heavy chains of tetanus neurotoxin and botulinum neurotoxin serotypes A, B and E as tools to study the neurospecific binding of the holotoxins. We found that the recombinant 50 kDa carboxy-terminal domains of tetanus and botulinum neurotoxins alone are responsible for the specific binding and internalisation into spinal cord cells in culture. Moreover, we provide evidence that the recombinant fragments block the internalization of the parental holotoxins in a dose-dependent manner, as determined by following the neurotoxin-dependent cleavage of their targets VAMP/synaptobrevin and SNAP-25. In addition, the recombinant binding fragments cause a significant delay in the paralysis induced by the corresponding holotoxin on the mouse phrenic nerve-hemidiaphragm preparation. Taken together, these results show that the carboxy-terminal domain of tetanus and botulinum neurotoxins is necessary and sufficient for the binding and internalisation of these proteins in neurons and open the possibility to use them as tools for the functional characterisation of the intracellular transport of clostridial neurotoxins.

Binding and Transcytosis of Botulinum Neurotoxin by Polarized Human Colon Carcinoma Cells

T-84 and Caco-2 human colon carcinoma cells and Madin-Darby canine kidney (MDCK) cells were used to study binding and transcytosis of iodinated Clostridium botulinum neurotoxin serotypes A, B, and C, as well as tetanus toxin. Specific binding and transcytosis were demonstrated for serotypes A and B in intestinal cells. Using serotype A as an example, the rate of transcytosis by T-84 cells was determined in both apical to basolateral (11.34 fmol/h/cm2) as well as basolateral to apical (8.98 fmol/h/cm2) directions, and by Caco-2 cells in the apical to basolateral (8.42 fmol/h/cm2) direction. Serotype A retained intact di-chain structure during transit through T-84 or Caco-2 cells, and when released on the basolateral side was toxic in vivo to mice and in vitro on mouse phrenic nerve-hemidiaphragm preparations. Serotype C and tetanus toxin did not bind effectively to T-84 cells, nor were they efficiently transcytosed (8-10% of serotype A). MDCK cells did not bind or efficiently transcytose (0.32 fmol/h/cm2) botulinum toxin. Further characterization demonstrated that the rate of transcytosis for serotype A in T-84 cells was increased 66% when vesicle sorting was disrupted by 5 microM brefeldin A, decreased 42% when microtubules were disrupted by 10 microM nocodazole, and decreased 74% at 18 degreesC. Drugs that antagonize toxin action at the nerve terminal, such as bafilomycin A1 (which prevents acidification of endosomes) and methylamine HCl (which neutralizes acidification of endosomes), produced only a modest inhibitory effect on the rate of transcytosis (17-22%). These results may provide an explanation for the mechanism by which botulinum toxin escapes the human gastrointestinal tract, and they may also explain why specific serotypes cause human disease and others do not.

Identification of phospholipase A 2 and neurotoxic activities in the venom of the New Guinean small-eyed snake ( Micropechis ikaheka)

The Papua New Guinean small-eyed snake ( Micropechis ikaheka) is recognised as a cause of life-threatening envenoming in certain parts of New Guinea. The clinical features suggest the presence of toxins acting at the neuromuscular junction and on muscle. We have used the mouse phrenic nerve-hemidiaphragm preparation, a phospholipase A 2 assay, and 125I-neurotoxin-binding radioimmunoassays to look for toxic activities in the crude venom and in preliminary high-performance liquid chromatography (HPLC) fractions. Micropechis ikaheka venom at 1 and 3 μg/ml completely abolished nerve-evoked muscle twitch within 70 min at 37 °C. There was also a sustained contracture of the muscle and some reduction in twitch tension evoked by direct stimulation; these were explained by the presence of phospholipase A 2 activity. The venom inhibited the binding of 125I-α-bungarotoxin to detergent-extracted human muscle acetylcholine receptor (AChR), and inhibited acetylcholine receptor function in a muscle cell line. It also inhibited binding of 125I-ω-conotoxin GVIA to detergent-extracted human frontal cortex voltage-gated calcium channels, but this appeared to be dependent on the phospholipase A 2 activity. Identification of the main neurotoxic fractions following HPLC are shown.

Blockade of neuromuscular transmission by huwentoxin-I, purified from the venom of the Chinese bird spider Selenocosmia huwena

Huwentoxin-I (HWTX-I) is a neurotoxic peptide purified from the venom of the Chinese bird spider Selenocosmia huwena. The effects of HWTX-I on neuromuscular transmission of vertebrate skeletal muscle have been investigated by means of twitch tension and electrophysiological techniques. On isolated mouse phrenic nerve-hemidiaphragm preparations, HWTX-I blocked the twitch responses to indirect, but not to direct, muscle stimulation. The time needed for complete block of the neuromuscular transmission was dose dependent. The transmission could be mostly restored by prolonged repeated washing with Tyrode's solution. If the preparation was pretreated with d-tubocurarine and then immersed in a mixed solution of d-tubocurarine and HWTX-I, the washout time necessary to restore the neuromuscular transmission was significantly decreased. Intracellular recording at the end-plate region of frog sartorius muscle revealed that HWTX-I could synchronously reduce the amplitude of the acetylcholine potential induced by ionophoretic application of acetylcholine as well as the amplitude of the end-plate potential evoked by nerve stimulation. Both of these effects eventually disappeared; however, both could be restored by prolonged washing. Experiments on Xenopus embryonic myocytes indicated that HWTX-I reduced the open probability of acetylcholine-induced channel activity, and finally blocked the channel. All of these results demonstrated that HWTX-I was a peptide neurotoxin and the postsynaptic nicotinic acetylcholine receptor was its site of action.

A study on the cause of death due to Waglerin-I, a toxin from Trimeresurus wagleri

Waglerin-I, a lethal toxin isolated from the venom of Trimeresurus wagleri, consists of 22 amino acid residues with a proline-rich sequence. In the present study, we investigated the cause of death and the possible site of action of this toxin. In anesthetized mice i.v. administration of waglerin-I at 0.5 μg/g produced respiratory failure within 5 min. The blood pressure could be maintained by the application of artificial respiration immediately after respiratory arrest. Waglerin-I at 4 μM reversibly blocked the indirect twitch of the mouse phrenic nerve-hemidiaphragm preparation, but had no effect on the direct twitch. It is concluded that respiratory failure, resulting from neuromuscular block, is the primary cause of death due to waglerin-I in mice. In contrast to the results obtained in mice, in anesthetized rats no toxic effects on respiration and blood pressure were observed except for a transient hypotension up to 10 μg/g (i.v.). In accordance with the in vivo experiments, waglerin-I at concentrations up to 40 μM did not block the indirect twitch of the rat diaphragm. It thus appears that rats are resistant to the toxic effects of waglerin-I.

Presynaptic A1-purinoceptor-mediated inhibitory effects of adenosine and its stable analogues on the mouse hemidiaphragm preparation

1. The effect of adenosine or its stable analogues (2-chloroadenosine, CADO; 5'-N-ethylcarboxamidoadenosine, NECA; and N6-cyclopentyladenosine, CPA) on the release of [3H]-acetylcholine ([3H]-ACh), and on the development of force of contraction evoked by electrical stimulation of the nerve, were studied in the mouse phrenic nerve-hemidiaphragm preparation. Evidence was obtained that the release of ACh is subject to presynaptic modulation through presynaptic A1(P1)-purinoceptors. 2. Adenosine or its stable analogues (CADO, NECA, CPA) inhibited, in a concentration-dependent manner, both the release of ACh and the force of the indirectly elicited contraction of hemidiaphragm preparation, provided in the latter case that the margin of safety was reduced by (+)-tubocurarine or magnesium. The order of potency in reducing ACh release was CPA greater than NECA greater than CADO greater than adenosine with IC50 values of 0.08 +/- 0.01, 0.74 +/- 0.05, 9.05 +/- 0.20, and 410.2 +/- 42.5 mumol/l, respectively. The order of potency in reducing twitch tension was CPA greater than NECA greater than CADO greater than adenosine with IC50 values of 0.11 +/- 0.02, 0.48 +/- 0.03, 2.07 +/- 0.49, and 240.4 +/- 20.0 mumol/l, respectively. 3. 8-Phenyltheophylline (8-PT) antagonized the inhibitory effects of the adenosine receptor agonists on ACh release and twitch tension.(ABSTRACT TRUNCATED AT 250 WORDS)

Dendrotoxin-like effects of noxiustoxin

Noxiustoxin from the Mexican scorpion (Centruroides noxius Hoffmann) is known to block neuronal K+ channels. Noxiustoxin facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations, but not in mouse phrenic nerve-hemidiaphragm preparations. Noxiustoxin displaced binding of a radiolabelled dendrotoxin from synaptosomal membranes from rat brain, with a Ki of 10−10M. It is concluded that noxiustoxin shares some pharmacological properties with the K+ channel blocking dendrotoxins.