Chick Biventer Cervicis Preparations Research Articles

Action of heparin and acetylcholine modulators on the neurotoxicity of the toad Rhinella schneideri (Anura: Bufonidae) in Brazil

Introduction: Rhinella schneideri is a toad widely distributed in South America and its poison is characterized by inducing cardiotoxicity and neurotoxicity. Objective: In this work, we investigated pharmacological strategies to attenuate the peripheral neurotoxicity induced by R. schneideri poison in avian neuromuscular preparation. Methods: The experiments were carried out using isolated chick biventer cervicis preparation subjected to field stimulation for muscle twitches recordings or exposed to acetylcholine and potassium chloride for contracture responses. Results: Poison (10 μg/ml) produced complete neuromuscular blockade in chick biventer cervicis preparation within approximately 70 min incubation (times for 50 and 90 % blockade: 15 ± 3 min and 40 ± 2 min, respectively; P < 0.05, N= 5); contracture responses to exogenous acetylcholine and KCl were unaffected by poison indicating no specificity with postsynaptic receptors or myotoxicity, respectively. Poison (10 μg/ml)-induced neuromuscular blockade was not prevented by heparin (5 and 150 IU/ml) under pre- or post-treatment conditions. Incubation at low temperature (23-25 °C) abolished the neuromuscular blockade; after raising the temperature to 37 °C, the complete neuromuscular blockade was slightly slower than that seen in preparations directly incubated at 37 °C (times for 50 and 90 % blockade: 23 ± 2 min and 60 ± 2.5 min, respectively; P < 0.05, N= 4). Neostigmine (3.3 μM) did not reverse the neuromuscular blockade in BC preparation whereas 3,4-diaminopyridine (91.6 μM) produced a partial and sustained reversal of the twitch responses (29 ± 7.8 % of maximal reversal reached in approximately 40 min incubation; P < 0.05, N= 4). Conclusions: R. schneideri poison induces potent peripheral neurotoxicity in vitro which can be partially reversible by 3,4-diaminopyridine.

Presynaptic Activity of an Isolated Fraction from Rhinella schneideri Poison.

Purpose: Rhinella schneideri is a toad found in many regions of the South America. The poison of the glands has cardiotoxic effect in animals and neuromuscular effects in mice and avian preparation. The purpose of this work was to identify the toxin responsible for the neuromuscular effect in avian and mice neuromuscular preparation.Methods: The methanolic extract from R. schneideri poison was fractioned by reversed phase HPLC. The purity and molecular mass were determined by LC/MS mass spectrometry. Chick biventer cervicis and mouse phrenic-nerve diaphragm were used as neuromuscular preparations to identify the toxin.Results: The purification resulted in 32 fractions, which 4 of them were active in neuromuscular preparation. The toxin of fraction 20 were chosen for better reproducibility of the whole extract activity and its molecular mass was 730.6 Da. The toxin produced facilitation of the muscle contraction followed by a complete neuromuscular blockade in chick biventer cervicis preparation in 90 min without interfering with the exogenous response to ACh and KCl. The quantal content was increased from 128 ± 13 (control) to 216 ± 44 (after 5 min and sustained until 60 min) in the presence of the toxin.Conclusion: In conclusion, our results demonstrated that the neuromuscular action of the poison of Rhinella schneideri is a multitoxin effect. More, the present work first isolated a 730.6 Da toxin that better represent the whole poison neuromuscular effect, to which is attributed a presynaptic action in avian and mouse neuromuscular preparation.

Mechanism of Rhinella icterica (Spix, 1824) toad poisoning using in vitro neurobiological preparations

The biological activity of Rhinella icterica toxic secretion (RITS) was evaluated on chick neuromuscular junctions, rat heart́s tissue and mice hippocampal slices. At chick biventer cervicis preparation, RITS (5, 10 and 20μg/mL) produced a concentration-independent irreversible neuromuscular blockade, which was preceded by a transitory increase of muscle twitch tension with the lowest concentration, in 120min recordings. In this set of experiments, RITS incubation partially prevented the curare neuromuscular blockade. The assessment of chick biventer cervicis muscle acetylcholinesterase (AChE) in the presence of RITS showed a significant inhibition of the enzyme, similarly to neostigmine. The incubation of muscles with digoxin or ouabain mimicked the poison activity by increasing the amplitude of the twitches followed by a progressive depression of the muscle strength. In addition, RITS demonstrated a digitalic-like activity, by inhibiting significantly the cardiac Na+, K+-ATPase. When the central nervous system was accessed, RITS induced an increase in the cell viability, in the lowest concentration. In addition, the poison protected slices subject to oxygen/glucose deprivation. Altogether, these data indicate that the poisonous extract of R. icterica is able to interfere with peripheral and central neurotransmission, probably due to a direct interaction with AChE, calcium channels and Na+, K+-ATPase. A further investigation upon the poison toxic components will unveil the components involved in such a pharmacological activity and the potential biotechnological application of this poison.

A survey on some biochemical and pharmacological activities of venom from two Colombian colubrid snakes, Erythrolamprus bizona (Double-banded coral snake mimic) and Pseudoboa neuwiedii (Neuwied's false boa)

Colombian colubrid snake venoms have been poorly studied. They represent a great resource of biological, ecological, toxinological and pharmacological research. We assessed some enzymatic properties and neuromuscular effects of Erythrolamprus bizona and Pseudoboa neuwiedii venoms from Colombia. Proteolytic, amidolytic and phospholipase A2 (PLA2) activities were analyzed using colorimetric assays and the neuromuscular activity was analyzed in chick biventer cervicis (BC) preparations. The venom of both species showed very low PLA2 and amidolytic activities; however, both exhibited high proteolytic activity, which in E. bizona venom surpassed that of P. neuwiedii venom. E. bizona and P. neuwiedii venoms provoked partial neuromuscular blockade, which was more prominent in P. neuwiedii venom. E. bizona venom (30 μg/ml) induced a significant potentiation of the contracture response to exogenous ACh (110 μM), which was not accompanied by twitch height alteration, whereas the highest venom concentration (100 μg/ml) inhibited contracture responses to both ACh and KCl (40 mM). In contrast, P. neuwiedii venom (30 and 100 μg/ml) caused significant reduction in the contracture responses to exogenous ACh and KCl. The morphological analyses showed high myotoxic effects in the muscle fibers of BC incubated with either venoms; however, they are more prominent in the P. neuwiedii venom. Our results suggest that the myotoxicity of the venom of the two Colombian species can be ascribed to their high proteolytic activity. An interesting data was the potentiation of the ACh-induced contracture, but not the twitch height, caused by E. bizona venom, at a concentration that is harmless to muscle fibers integrity. This phenomenon remains to be further elucidated, and suggest that a possible involvement of post-synaptic receptors cannot be discarded. This work is a contribution to expand the knowledge on colubrid venoms; it allows envisaging that the two venoms offer the potential to go further in the identification of their components and biological targets.

Neutralising ability of Terminalia fagifolia extract (Combretaceae) against the in vitro neuromuscular effects of Bothrops jararacussu venom

The ability of Terminalia fagifolia hydroalcoholic extract (Tf-HE) to neutralise the paralysis and myotoxicity induced by Bothrops jararacussu venom was assayed using mouse phrenic nerve-diaphragm (PND) preparation and two varieties of chick biventer cervicis (BC) preparations. Tf-HE 100 μg/mL and 500 μg/mL were tested against 40 and 200 μg of venom/mL in PND and BC preparations, respectively, using pre- and post-venom incubation treatments. The effects of Tf-HE against the myotoxicity caused by venom were evaluated via histological analysis (PND) and creatine kinase (CK) release (BC). Tf-HE was able to reverse the venom paralysis in both preparation types. The contractures to exogenous ACh in BC preparations showed that Tf-HE may act on extrinsic, preserving those intrinsic postsynaptic receptors. There was a positive correlation between CK and morphological changes. The high non-hemolytic saponin content can explain the Tf-HE efficacy against the toxic effects of B. jararacussu venom in vertebrate neuromuscular preparations.

Exploring and understanding the functional role, and biochemical and structural characteristics of an acidic phospholipase A2, AplTx-I, purified from Agkistrodon piscivorus leucostoma snake venom

Phospholipases A2 (PLA2s) constitute a class of extensively studied toxins, isolated from snake venoms. Basic PLA2 isoforms mediate various toxicological effects, while the acidic isoforms generally have higher enzymatic activities, but do not promote evident toxic effects. The functions of these acidic isoforms in snake venoms are still not completely understood and more studies are needed to characterize the biological functions and diversification of acidic toxins in order to justify their abundant presence in these secretions. Recently, Lomonte and collaborators demonstrated, in a proteomic and toxicological study, high concentrations of PLA2s in the venom of Agkistrodon piscivorus leucostoma. We have, herein, purified and characterized an acidic PLA2 from this snake venom, denominated AplTx-I, in order to better understand its biochemical and structural characteristics, as well as its biological effects. AplTx-I was purified using two chromatographic steps, in association with enzymatic and biological assays. The acidic toxin was found to be one of the most abundant proteins in the venom of A. p. leucostoma; the protein was monomeric with a molecular mass of 13,885.8 Da, as identified by mass spectrometry ESI-TOF and electrophoresis. The toxin has similar primary and tridimensional structures to those of other acidic PLA2s, a theoretical and experimental isoelectric point of ≈5.12, and a calcium-dependent enzyme activity of 25.8985 nM/min/mg, with maximum values at 37 °C and pH 8.0. Despite its high enzymatic activity on synthetic substrate, AplTx-I did not induce high or significant myotoxic, coagulant, anticoagulant, edema, neuromuscular toxicity in mouse phrenic nerve-diaphragm preparations or antibacterial activities. Interestingly, AplTx-I triggered a high and selective neuromuscular toxicity in chick biventer cervicis preparations. These findings are relevant to provide a deeper understanding of the pharmacology, role and diversification of acidic phospholipase A2 isoforms in snake venoms.

Cross-Neutralisation of In Vitro Neurotoxicity of Asian and Australian Snake Neurotoxins and Venoms by Different Antivenoms.

There is limited information on the cross-neutralisation of neurotoxic venoms with antivenoms. Cross-neutralisation of the in vitro neurotoxicity of four Asian and four Australian snake venoms, four post-synaptic neurotoxins (α-bungarotoxin, α-elapitoxin-Nk2a, α-elapitoxin-Ppr1 and α-scutoxin; 100 nM) and one pre-synaptic neurotoxin (taipoxin; 100 nM) was studied with five antivenoms: Thai cobra antivenom (TCAV), death adder antivenom (DAAV), Thai neuro polyvalent antivenom (TNPAV), Indian Polyvalent antivenom (IPAV) and Australian polyvalent antivenom (APAV). The chick biventer cervicis nerve-muscle preparation was used for this study. Antivenom was added to the organ bath 20 min prior to venom. Pre- and post-synaptic neurotoxicity of Bungarus caeruleus and Bungarus fasciatus venoms was neutralised by all antivenoms except TCAV, which did not neutralise pre-synaptic activity. Post-synaptic neurotoxicity of Ophiophagus hannah was neutralised by all antivenoms, and Naja kaouthia by all antivenoms except IPAV. Pre- and post-synaptic neurotoxicity of Notechis scutatus was neutralised by all antivenoms, except TCAV, which only partially neutralised pre-synaptic activity. Pre- and post-synaptic neurotoxicity of Oxyuranus scutellatus was neutralised by TNPAV and APAV, but TCAV and IPAV only neutralised post-synaptic neurotoxicity. Post-synaptic neurotoxicity of Acanthophis antarcticus was neutralised by all antivenoms except IPAV. Pseudonaja textillis post-synaptic neurotoxicity was only neutralised by APAV. The α-neurotoxins were neutralised by TNPAV and APAV, and taipoxin by all antivenoms except IPAV. Antivenoms raised against venoms with post-synaptic neurotoxic activity (TCAV) cross-neutralised the post-synaptic activity of multiple snake venoms. Antivenoms raised against pre- and post-synaptic neurotoxic venoms (TNPAV, IPAV, APAV) cross-neutralised both activities of Asian and Australian venoms. While acknowledging the limitations of adding antivenom prior to venom in an in vitro preparation, cross-neutralization of neurotoxicity means that antivenoms from one region may be effective in other regions which do not have effective antivenoms. TCAV only neutralized post-synaptic neurotoxicity and is potentially useful in distinguishing pre-synaptic and post-synaptic effects in the chick biventer cervicis preparation.

Biological activities of Leptodeira annulata (banded cat-eyed snake) venom on vertebrate neuromuscular preparations

The physiological properties of colubrid snake venoms are largely unknown and less frequently investigated. In this study, we assessed the enzymatic properties and biological activities of Leptodeira annulata (banded cat-eyed snake) venom, an opistoglyphous snake from Colombia. The proteolytic, phospholipase A2 and amidolytic activities are assessed using colorimetric assays and the biological activities were analyzed in avian and mammalian neuromuscular preparations. L. annulata venom caused neuromuscular blockade in chick biventer cervicis (BC) preparations (40± 15% and 50± 3% of twitch reduction for 30 and 100 μg/ml, respectively; p < 0.05) following 120 incubation; 10 μg/ml of venom did not induce blockade. There was a mild reduction in contracture response to exogenous acetylcholine (110 μM) in BC preparations exposed to 10 and 30 μg of venom/ml (∼4% and ∼32% of reduction, respectively, p > 0.05, n = 4) compared to basal values whereas the highest concentration (100 μg/ml) abolished it after 120 min. The venom caused a significant reduction in contracture response elicited by KCl (∼58 and ∼90 of reduction for 30 and 100 μg/ml, respectively, p < 0.05, n = 4). In mouse phrenic nerve-diaphragm (PND) preparations, L. annulata venom induced a progressive muscle membrane depolarization [from −85.9 ± 1.6 mV (t0) to −72.2 ± 2.9 mV (t120), p < 0.05, n = 4); the postsynaptic receptors remained functional as shown by carbachol-induced depolarization. The morphological analyses showed a concentration-dependent number of pathological states in muscle fibers from both BC and PND preparations pre-exposed to venom. The venom showed high proteolytic activity and low phospholipase A2 activity; there was no evidence for serine protease activity. These results indicate that the neuromuscular effect induced by L. annulata venom resulted from damaged muscle fibers that lead to the blockade of twitches response. The findings suggest that the myotoxicity might be related to the presence of metalloproteases in this venom.

Presynaptic Proteins as Markers of the Neurotoxic Activity of BmjeTX-I and BmjeTX-II Toxins from Bothrops marajoensis (Marajó Lancehead) Snake Venom.

Neuromuscular preparations exposed to B. marajoensis venom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release (synaptophysin, synaptobrevin, and SNAP25 proteins), the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposed in vitro to BmjeTX-I and BmjeTX-II toxins from B. marajoensis venom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.

Neuromuscular Activity of Micrurus laticollaris (Squamata: Elapidae) Venom in Vitro

In this work, we have examined the neuromuscular activity of Micrurus laticollaris (Mexican coral snake) venom (MLV) in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations, the MLV induced an irreversible concentration- and time-dependent (1–30 µg/mL) neuromuscular blockade, with 50% blockade occurring between 8 and 30 min. Muscle contractures evoked by exogenous acetylcholine were completely abolished by MLV, whereas those of KCl were also significantly altered (86% ± 11%, 53% ± 11%, 89% ± 5% and 89% ± 7% for one, three, 10 and 30 µg of venom/mL, respectively; n = 4; p < 0.05). In mouse phrenic nerve-diaphragm preparations, MLV (1–10 µg/mL) promoted a slight increase in the amplitude of twitch-tension (3 µg/mL), followed by neuromuscular blockade (n = 4); the highest concentration caused complete inhibition of the twitches (time for 50% blockade = 26 ± 3 min), without exhibiting a previous neuromuscular facilitation. The venom (3 µg/mL) induced a biphasic modulation in the frequency of miniature end-plate potentials (MEPPs)/min, causing a significant increase after 15 min, followed by a decrease after 60 min (from 17 ± 1.4 (basal) to 28 ± 2.5 (t15) and 12 ± 2 (t60)). The membrane resting potential of mouse diaphragm preparations pre-exposed or not to d-tubocurarine (5 µg/mL) was also significantly less negative with MLV (10 µg/mL). Together, these results indicate that M. laticollaris venom induces neuromuscular blockade by a combination of pre- and post-synaptic activities.

Biochemical Characterization of a PLA2 Btae TX-I Isolated from Bothriopsis taeniata Snake Venom: A Pharmacological and Morphological Study

In this research a preliminary identification and biochemical and biological characterization of a PLA2 (Btae TX-I) from the venom of a viperid snake, Bothriopsis taeniata (Speckled forest pit viper) were obtained. Btae TX-I was purified by two chromatographic steps, molecular exclusion chromatography followed by analytical chromatography reverse phase HPLC. Molecular mass behaved as a homogeneous single chain protein on SDS–PAGE, confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 13889.98 Da. Tryptic peptides were determined in tandem mass spectrometry and showed similarity with other myotoxic PLA2s. Btae TX-I belongs to the Asp49 PLA2 class, is enzymatically active in presence of a synthetic substrate and shows a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35–45°C. PLA2 activity in presence of Mn2+, Mg2+, Cd2+ and Zn2+ was reduced either in presence or absence of Ca2+, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca2+. Crotalic crotapotins from rattlesnake venom has significantly inhibited (p<0.05) the enzymatic activity of Btae TX-I. In ex vivo experiment, Btae TX-I caused partial blockade of the neuromuscular transmission in chick biventer cervicis preparations in a similar way to other Bothrops species. Btae TX-I also inhibited contractures in the upper concentration (50 μg) to exogenous KCl (20 mM). Histological analysis of the biventer cervicis incubated with Btae TX-I showed that just the highest Btae TX-I PLA2 dose (50 μg) caused almost 27.4 ± 0.3% damaged fibers. The results give evidence that the main effect of type Asp49 Btae TX-I PLA2 from Bothriopsis taeniata is at the post-synaptic site.

Evidence of presynaptic and postsynaptic action of local anesthetics in rats

To assess the probable actions of ropivacaine, 50% enantiomeric excess bupivacaine mixture (S75-R25) and levobupivacaine on neuromuscular transmission in vitro. Thirty rats were distributed into groups (n=5) according to the drug used: ropivacaine, bupivacaine (S75-R25) and levobupivacaine. The concentration used for the three local anesthetics (LA) was 5 µg.mL. (-1)The following parameters were evaluated: 1) LA effects on membrane potential (MP) and miniature end plate potential (MEPP). A chick biventer cervicis preparation was also used to evaluate LA effects on the contracture response to acetylcholine. LA did not alter MP values and decreased the frequency and amplitude of MEPP. In a chick biventer cervicis preparation, bupivacaine (S75-R25) and levobupivacaine decreased the contracture response to acetylcholine with statistical significance, in comparison to ropivacaine. In the concentrations used, levobupivacaine and bupivacaine (S75-R25) exhibited presynaptic and postsynaptic actions evidenced by alterations in miniature end plate potentials and contracture response to acetylcholine. Ropivacaine only had a presynaptic action.

VdTX-1, a reversible nicotinic receptor antagonist isolated from venom of the spider Vitalius dubius (Theraphosidae)

Theraphosid spider venoms can block neurotransmission in vertebrate nerve–muscle preparations in vitro, but few of the components involved have been characterized. In this work, we describe the neuromuscular activity of venom from the Brazilian theraphosid Vitalius dubius and report the purification and pharmacological characterization of VdTX-1, a 728 Da toxin that blocks nicotinic receptors. Neuromuscular activity was assayed in chick biventer cervicis preparations and muscle responses to exogenous ACh and KCl were determined before and after incubation with venom or toxin. Changes in membrane resting potential were studied in mouse diaphragm muscle. The toxin was purified by a combination of filtration through Amicon® filters, cation exchange HPLC and RP-HPLC; toxin purity and mass were confirmed by mass spectrometry. Venom caused progressive neuromuscular blockade and muscle contracture; the blockade but not the contracture was reversible by washing. Venom attenuated contractures to exogenous ACh and KCl. Filtration yielded low (LM, <5 kDa) and high (HM, >5 kDa) fractions, with the latter reproducing the contracture seen in venom but with a slight and progressive twitch blockade. The LM fraction caused reversible blockade and attenuated contractures to ACh, but had no effect on contractures to KCl. VdTX-1 (728 Da) purified from the LM fraction was photosensitive and reduced the Emax to ACh in biventer cervicis muscle without affecting the EC50; VdTX-1 also abolished carbachol-induced depolarizations. V. dubius venom contains at least two components that affect vertebrate neurotransmission. One component, VdTX-1, blocks nicotinic receptors non-competitively to produce reversible blockade without muscle contracture.

Protection by Mikania laevigata (guaco) extract against the toxicity of Philodryas olfersii snake venom

Philodryas olfersii is responsible for most colubrid snakebites in Brazil. In this work, we examined the ability of an ethanolic extract from Mikania laevigata (guaco) leaves to protect against the in vitro neuromuscular activity of P. olfersii venom in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) preparations. M. laevigata extract caused moderate twitch-tension facilitation at low concentrations (107.4 ± 6.2% with 20 μl/ml and 118.9 ± 9.3% with 40 μl/ml in PND, and 120.7 ± 7.7% with 40 μl/ml and 114.5 ± 4.4% with 50 μl/ml in BC after 120 min; n = 4–6, mean ± SEM). In PND, the ethanol alone (40 μl/ml, n = 4) did not change the twitch-tension when compared with control. However, in BC, the ethanol produced a higher facilitation when compared to control. At higher concentrations (>50 μl/ml) the extract caused total and reversible blockade in both preparations. Venom (50 μg/ml) caused partial blockade in PND (58.5 ± 12%, n = 4) and almost total blockade in BC (93.5 ± 2.2%, n = 4). Pretreatment of the preparations with extract (40 μl/ml) for 30 min before incubation with venom (50 μg/ml) completely protected PND from neuromuscular blockade and delayed the blockade in BC. The extract alone caused only mild morphological alterations (12.5 ± 0.5% and 10.9 ± 2.3% fiber damage in PND and BC, respectively, compared to 2.3 ± 0.3% and 3 ± 0 in controls; n = 3), with no increase in expression of the inflammatory cytokines TNFα and IFNγ. The ethanol alone also caused slight muscle damage: 4.3 ± 2.4% in PND and 6.7 ± 3.3% in BC (both n = 3) and little or no TNFα and IFNγ expression in both preparations as observed in control. Venom (50 μg/ml) caused 53.5 ± 8.5% and 55.8 ± 4.3% fiber damage in PND and BC, respectively; (n = 3, p < 0.05 vs. controls) and enhanced expression of TNFα and IFNγ. Pretreatment of the preparations with extract protected against venom-induced muscle damage by 80.3 and 60.4 in PND and BC, respectively, and prevented TNFα and IFNγ expression. These results indicate that the M. laevigata extract protected nerve-muscle preparations against the myotoxic, neurotoxic and inflammatory effects of P. olfersii venom.

Pharmacological and partial biochemical characterization of Bmaj-9 isolated from Bothrops marajoensis snake venom

Bmaj-9, a basic PLA2 (13679.33 Da), was isolated from Bothrops marajoensis snake venom through only one chromatographic step in reversed phase HPLC on ¼-Bondapak C-18 column. The amino acid composition showed that Bmaj-9 had a high content of Lys, His, and Arg, typical of a basic PLA2. The sequence of Bmaj-9 contains 124 amino acid residues with a pI value of 8.55, such as DLWQWGQMIL KETGKLPFSY YTAYGCYCGW GGRGGKPKAD TDRCCFVHDC, revealing a high homology with Asp49 PLA2 from other snake venoms. It also exhibited a pronounced phospholipase A2 activity when compared with crude venom. In chick biventer cervicis preparations, the time for 50% and 100% neuromuscular paralysis was respectively (in minutes): 110 ± 10 (1 µg/mL); 40 ± 6 and 90 ± 2 (5 µg/mL); 30 ± 3 and 70 ± 5 (10 µg/mL); 42 ± 1 and 60 ± 2 (20 µg/mL), with no effect on the contractures elicited by either exogenous ACh (110 µM) or KCl (20 mM). Bmaj-9 (10 µg/mL) neither interfered with the muscular response to direct electrical stimulation in curarized preparations nor significantly altered the release of CK at 0, 15, 30 and 60 minutes incubations (27.4 ± 5, 74.2 ± 8, 161.0 ± 21 and 353.0 ± 47, respectively). The histological analysis showed that, even causing blockade at the maximum dosage (5 µg/mL), the toxin does not induce significant morphological alterations such as necrosis or infiltration of inflammatory cells. These results identified Bmaj-9 as a new member of the basic Asp49 PLA2 family able to interact with the motor nerve terminal membrane, thereby inducing a presynaptic neuromuscular blockade.

Presynaptic action of Bothriopsis bilineata smargadina (forest viper) venom in vitro

In this work, we examined the neuromuscular activity of Bothriopsis bilineata smargadina (forest viper) venom in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations the venom caused concentration-dependent (0.1–30μg/ml) neuromuscular blockade that was not reversed by washing, with 50% blockade occurring in 15–90min. Muscle contractures to exogenous acetylcholine and KCl were unaffected by venom, but there was a slight increase in creatine kinase release after 120min (from 80±15 to 206±25U/ml, n=6, p<0.05). In mouse phrenic nerve-diaphragm preparations, the venom (1, 10 and 30μg/ml) produced marked facilitation (∼120% increase above basal) at the highest concentration followed by neuromuscular blockade; the effects at lower concentrations were considerably less marked. Venom increased the quantal content values after 15 and 30min followed by significant inhibition at ≥90min. However, venom did not alter the muscle membrane resting potential or the response to exogenous carbachol. In both preparations, incubation at 22°C instead of 37°C delayed the onset of blockade, as did inhibition of venom PLA2 activity. In curarized mouse preparations, the venom produced only muscle facilitation. These results indicate that B. b. smargadina venom causes neuromuscular blockade in vitro by a presynaptic mechanism involving PLA2.

Some actions of 4,17a-dimethyl-4,17a-diaza-D-homo-5alpha-androstane dimethiodide (HS-342), a new neuromuscular blocking drug.

Abstract The effects of the newly-synthesized bisquaternary steroid 4,17a-dimethyl-4,17a-diaza-D-homo-5α-androstane dimethiodide (HS-342) have been investigated on the rat isolated phrenic nerve-hemidiaphragm, chick biventer cervicis and intact cat tibialis anterior muscle preparations, on the guinea-pig isolated ileum and on the rat isolated vas deferens preparations. The anticholinesterase activity of HS-342 was also measured. In the skeletal muscle preparations HS-342 exhibited properties of a post-junctionally active neuromuscular blocking agent of the non-depolarizing class. In the anaesthetized cat HS-342 exhibited approximately equal activity to that of tubocurarine, but its duration of action was about one-third of that of tubocurarine. HS-342 exhibited low anticholinesterase activity, low muscarinic blocking activity and apparently had no effect on the adrenergic neuroeffector junction studied.

Biochemical and pharmacological characterization of PhTX-I a new myotoxic phospholipase A2 isolated from Porthidium hyoprora snake venom

This paper reports the biochemical and pharmacological characterization of a new myotoxic PLA2 (EC 3.1.1.4) called PhTX-I, purified from Porthidium hyoprora venom by one step analytical chromatography reverse phase HPLC. The homogeneity of the PhTX-I fraction and its molecular mass were initially evaluated by SDS–PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14.249Da and constituted of a single polipeptidic chain. Amino acid sequence was determined by “de novo sequencing,” in tandem mass spectrometry, belonging to D49-PLA2 enzyme class and exhibiting high identity (44–90%) with other myotoxics PLA2 from snake venoms. The enzymatic investigation showed maximal activity at pH 8 and 35–45°C. This activity was dependent on Ca2+, other cations (Mg2+, Mn2+, Cd2+ and Zn2+) reduced notably the enzymatic activity, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca2+. Ex vivo, whole venom and PhTX-I PLA2 caused blockade of the neuromuscular transmission in young chick biventer cervicis preparations similar to other isolated snake venom toxins from the Bothrops genus. In vivo, both induced local myotoxicity and systemic interleukin-6 response upon intramuscular injection, additionally, induced moderate footpad edema. In vitro, both induced low cytotoxicity in skeletal muscle myoblasts, however PhTX-I PLA2 was able to lyse myotubes.

Study of the reversal effect of NF449 on neuromuscular blockade induced by d-tubocurarine

AimsThe aim of this study was to investigate the mechanism for the reversal effect of NF449 (a suramin analogue) on the neuromuscular block induced by d-tubocurarine (d-TC). Main methodsNerve-stimulated muscle contractions and end-plate potentials were performed in mouse phrenic nerve-diaphragm preparations. Acetylcholine (ACh)-induced muscle contractions were performed in the chick biventer cervicis preparations. Presynaptic nerve terminal waveform recordings were performed in mouse triangularis sterni preparations. Key findingsAmongst the suramin analogues in this study, only the NF449 and suramin were able to reverse the blockade effect produced by d-TC on nerve-stimulated muscle contractions. Each of these suramin analogues (NF007, NF023, NF279 and NF449) alone has no significant effect on the amplitude of nerve-stimulated muscle contractions. NF449 and suramin also showed the antagonising effects on the inhibition of end-plate potentials induced by d-TC. Furthermore, pre-treatment with NF449 can antagonise the inhibition of d-TC in ACh-induced contractions of chick biventer cervicis muscle. NF449 produced a greater rightward shift of the dose–response inhibition curve for d-TC than did suramin. Because other purinergic 2X (P2X) receptor antagonists, NF023 and NF279, do not have the reverse effects on the neuromuscular blockade of d-TC, the effect of NF449 seems irrelevant to inhibition of P2X receptors. SignificanceThese data suggest that NF449 was able to compete with the binding of d-TC on the nicotinic ACh receptors, and the effect of NF449 was more potent than suramin in reducing the inhibition of d-TC. The structure of NF449 may provide useful information for designing potent antidotes against neuromuscular toxins.

Structural and pharmacological characterization of the crotamine isoforms III-4 (MYX4_CROCu) and III-7 (MYX7_CROCu) isolated from the Crotalus durissus cumanensis venom

Two major crotamine isoforms (III-4 and III-7) were obtained combining two chromatographic steps on molecular exclusion chromatography (Sephadex G-75) and ion-exchange column (Protein Pack SP 5PW) of the rattlesnake Crotalus durissus cumanensis venom. The “in vivo” myotoxic effect of the venom, its “in vitro” cytotoxicity in myoblasts and myotubes (C2C12) and the neurotoxic and edema-forming activity were characterized. The molecular masses of the crotamine isoforms were 4907.94 Da (III-4) and 4985.02 Da (III-7) and, as determined by mass spectrometry, both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing through tandem mass spectrometry was used to determine the primary structure of both isoforms. III-4 and III-7 isoforms presented a 42-amino acid residues sequence and showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. In vivo, both crotamine isoforms induced myotoxicty and a systemic interleukin-6 response upon intramuscular injection. These new crotamine isoforms induced low cytotoxicity in skeletal muscle myoblasts and myotubes (C2C12) and both induced a facilitatory effect on neuromuscular transmission in young chick biventer cervicis preparation. Edema-forming activity was also analyzed by injection of the crotamine isoforms into the right paw, since both crotamine isoforms exert a strong pro-inflammatory effect.