Action of heparin and acetylcholine modulators on the neurotoxicity of the toad Rhinella schneideri (Anura: Bufonidae) in Brazil / Acción de la heparina y moduladores de acetilcolina en la neurotoxicidad del sapo Rhinella schneideri (Anura: Bufonidae) en Brasil

Abstract 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.

Resumen Introducción: Rhinella schneideri está ampliamente distribuida en Suramérica y su veneno es caracterizado por inducir cardiotoxicidad y neurotoxicidad. Objetivo: En este trabajo, investigamos estrategias farmacológicas para atenuar la neurotoxicidad periférica inducida por el veneno de R. schneideri en preparaciones neuromusculares de aves. Métodos: Los experimentos fueron realizados usando preparaciones de biventer cervicis de pollos sometidas a estimulación de campo para el registro de las contracciones musculares o expuestas a la acetilcolina y al cloruro de potasio para la respuesta contractural. Resultados: El veneno (10 µg/ml) provocó un bloqueo neuromuscular completo en las preparaciones después de aproximadamente 70 min de incubación (tiempos para 50 y 90 % de bloqueo: 15 ± 3 min y 40 ± 2 min, respectivamente; P < 0.05, N = 5); las contracturas en respuesta a la acetilcolina y el KCl exógenos no fueron afectadas por el veneno, indicando que no hay una interacción especifica con receptores postsinápticos o miotoxicidad respectivamente. El bloqueo neuromuscular causado por el veneno (10 µg/ml) no fue prevenido por la heparina (5 y 150 UI/ml) bajo condiciones pre y post-tratamiento. La incubación a bajas temperaturas (23-25 ºC) abolió el bloqueo neuromuscular; después de aumentar la temperatura a 37 ºC, el bloqueo neuromuscular total fue levemente más lento que el visto en preparaciones directamente incubadas a 37 ºC (tiempos para 50 y 90 % de bloqueo: 23 ± 2 min y 60 ± 2.5 min, respectivamente; P < 0.05, N= 4). Neostigmina (3.3 µM) no revirtió el bloqueo neuromuscular, mientras que 3.4-diaminopiridina (91.6 µM) produjo una reversión parcial y sostenida de las respuestas neuromusculares (29 ± 7.8 % de la reversión máxima alcanzada en aproximadamente 40 min de incubación; P < 0.05, N = 4). Conclusiones: El veneno de R. schneideri indujo neurotoxicidad periférica potente in vitro, el cual puede ser revertido por 3.4-diaminopiridina.

Chemical and functional analyses of Rhinella icterica (Spix, 1824) toad secretion screened on contractions of the heart and oviduct in Locusta migratoria.

Rhinella icterica is a Brazilian toad with a parotoid secretion that is toxic to insects. In this work, we examined the entomotoxicity of this secretion in locust (Locusta migratoria) semi-isolated heart and oviduct preparations in vitro. The parotoid secretion caused negative chronotropism in semi-isolated heart preparations (at the highest dose tested: 500 µg) and markedly enhanced the amplitude of spontaneous contractions and tonus of oviduct muscle (0.001-100 µg). In addition, the secretion enhanced neurally-evoked contractions of oviduct muscle, which was more sensitive to low concentrations of secretion than the semi-isolated heart. The highest dose of secretion (100 µg) caused neuromuscular blockade. In zero calcium-high magnesium saline, the secretion still enhanced muscle tonus, suggesting the release of intracellular calcium to stimulate contraction. Reverse-phase HPLC of the secretion yielded eight fractions, of which only fractions 4 and 5 affected oviduct muscle tonus and neurally-evoked contractions. No phospholipase A2 activity was detected in the secretion or its chromatographic fractions. The analysis of fractions 4 and 5 by LC-DAD-MS/MS revealed the following chemical compounds: suberoyl arginine, hellebrigenin, hellebrigenin 3-suberoyl arginine ester, marinobufagin 3-pimeloyl arginine ester, telocinobufagin 3-suberoyl arginine ester, marinobufagin 3-suberoyl arginine ester, bufalin 3-adipoyl arginine, marinobufagin, bufotalinin, and bufalitoxin. These findings indicate that R. icterica parotoid secretion is active in both of the preparations examined, with the activity in oviduct possibly being mediated by bufadienolides.

Cordia salicifolia and Lafoensia pacari plant extracts against the local effects of Bothrops jararacussu and Philodryas olfersii snake venoms.

Philodryas olfersii produces similar local effects to Bothrops jararacussu snakebite, which can induce misidentification and bothropic antivenom administration. Antivenom therapy is effective, but has its limitations regarding local damage. Since plants are used in folk medicine to treat snakebite victims, we evaluated the protective properties of Cordia salicifolia and Lafoensia pacari extracts against Philodryas olfersii and Bothrops jararacussu venoms. Preparations pretreated with both extracts inhibited > 90% the B. jararacussu venom-induced neuromuscular blockade, and 52% to 81% the P. olfersii venom-induced blockade. C. salicifolia inhibited the myonecrosis promoted by both venoms; however, L. pacari prevented only the myofilaments hypercontraction. Regarding haemorrhagic activity, C. salicifolia was more effective against B. jararacussu venom, while L. pacari was more effective against P. olfersii venom. On the other hand, for oedema-forming activity the results were the opposite. Considering that both extracts prevented (to different levels) the main manifestations of both snakebites (local symptoms), we endorse further studies involving these plants as coadjuvant in snakebite therapeutics.

Chemical and Pharmacological Screening of Rhinella icterica (Spix 1824) Toad Parotoid Secretion in Avian Preparations.

The biological activity of Rhinella icterica parotoid secretion (RIPS) and some of its chromatographic fractions (RI18, RI19, RI23, and RI24) was evaluated in the current study. Mass spectrometry of these fractions indicated the presence of sarmentogenin, argentinogenin, (5ß,12ß)-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide, marinobufagin, bufogenin B, 11α,19-dihydroxy-telocinobufagin, bufotalin, monohydroxylbufotalin, 19-oxo-cinobufagin, 3α,12ß,25,26-tetrahydroxy-7-oxo-5ß-cholestane-26-O-sulfate, and cinobufagin-3-hemisuberate that were identified as alkaloid and steroid compounds, in addition to marinoic acid and N-methyl-5-hydroxy-tryptamine. In chick brain slices, all fractions caused a slight decrease in cell viability, as also seen with the highest concentration of RIPS tested. In chick biventer cervicis neuromuscular preparations, RIPS and all four fractions significantly inhibited junctional acetylcholinesterase (AChE) activity. In this preparation, only fraction RI23 completely mimicked the pharmacological profile of RIPS, which included a transient facilitation in the amplitude of muscle twitches followed by progressive and complete neuromuscular blockade. Mass spectrometric analysis showed that RI23 consisted predominantly of bufogenins, a class of steroidal compounds known for their cardiotonic activity mediated by a digoxin- or ouabain-like action and the blockade of voltage-dependent L-type calcium channels. These findings indicate that the pharmacological activities of RI23 (and RIPS) are probably mediated by: (1) inhibition of AChE activity that increases the junctional content of Ach; (2) inhibition of neuronal Na+/K+-ATPase, leading to facilitation followed by neuromuscular blockade; and (3) blockade of voltage-dependent Ca2+ channels, leading to stabilization of the motor endplate membrane.

Jaburetox, a natural insecticide derived from Jack Bean Urease, activates voltage-gated sodium channels to modulate insect behavior.

Jaburetox (Jbtx) is an insecticidal peptide derived from Canavalia ensiformis urease, whose mechanism of action is not completely elucidated. We employed behavioral, electromyographical and electrophysiological protocols to identify the cellular and molecular targets involved in the Jbtx entomotoxicity in cockroaches and locusts. In Nauphoeta cinerea, Jbtx (32 µg/g) altered the locomotory behaviour inducing a significative decrease in the distance travelled followed by a significant increase in stopped time (52 ±â€¯85 cm and 2573 ±â€¯89 s, p < .05, n = 40). Jbtx (8 to 32 µg/g body weight, respectively) also increased the leg and antennae grooming activities (p < .05, n = 40, respectively). Jbtx (8 to 16 µg/g) induced a maximum neuromuscular blockade of 80.72% (n = 6, p < .05) and was cardiotoxic, decreasing the cockroach heart rate. The electrophysiological profiles of both muscle and nerve of L. migratoria showed that Jbtx (2.5 × 10-7 and 2.5 × 10-3 µg/ body weight) induced a significant increase in the amplitude of nerve action potentials (n = 5, p < .05). Voltage clamp analysis of Jbtx (200 nM) applied in Xenopus laevis oocytes heterologously expressed with Nav 1.1 channels showed a significant increase in the sodium currents. In conclusion, this work revealed that the entomotoxic activity of Jbtx involves complex behavioral alterations that begins with an initial activation of voltage-gated sodium channels.

Jack bean urease modulates neurotransmitter release at insect neuromuscular junctions.

BACKGROUND: Plants have developed a vast range of mechanisms to compete with phytophagous insects, including entomotoxic proteins such as ureases. The legume Canavalia ensiformis produces several urease isoforms, of which the more abundant is called Jack Bean Urease (JBU). Previews work has demonstrated the potential insecticidal effects of JBU, by mechanisms so far not entirely elucidated. In this work, we investigated the mechanisms involved in the JBU-induced activity upon neurotransmitter release on insect neuromuscular junctions. METHODS: Electrophysiological recordings of nerve and muscle action potentials, and calcium imaging bioassays were employed. RESULTS AND CONCLUSION: JBU (0.28 mg/animal/day) in Locusta migratoria 2nd instar through feeding and injection did not induce lethality, although it did result in a reduction of 20% in the weight gain at the end of 168 h (n = 9, p ≤ 0.05). JBU (0.014 and 0.14 mg) injected direct into the locust hind leg induced a dose and time-dependent decrease in the amplitude of muscle action potentials, with a maximum decrease of 70% in the amplitude at the highest dose (n = 5, p ≤ 0.05). At the same doses JBU did not alter the amplitude of action potentials evoked from motor neurons. Using Drosophila 3rd instar larvae neuromuscular preparations, JBU (10-7 M) increased the occurrence of miniature Excitatory Junctional Potentials (mEJPs) in the presence of 1 mM CaCl2 (n = 5, p ≤ 0.05). In low calcium (0.4 mM) assays, JBU (10-7 M) was not able to modulate the occurrence of the events. In Ca2+-free conditions, with EGTA or CoCl2, JBU induced a significant decrease in the occurrence of mEPJs (n = 5, p ≤ 0.05). Injected into the 3rd abdominal ganglion of Nauphoeta cinerea cockroaches, JBU (1 µM) induced a significant increase in Ca2+ influx (n = 7, p ≤ 0.01), similar to that seen for high KCl (35 mM) condition. Taken together the results confirm a direct action of JBU upon insect neuromuscular junctions and possibly central synapses, probably by disrupting the calcium machinery in the pre-synaptic region of the neurons.

Methylprednisolone as a memory enhancer in rats: Effects on aversive memory, long-term potentiation and calcium influx.

It is well recognized that stress or glucocorticoids hormones treatment can modulate memory performance in both directions, either impairing or enhancing it. Despite the high number of studies aiming at explaining the effects of glucocorticoids on memory, this has not yet been completely elucidated. Here, we demonstrate that a low daily dose of methylprednisolone (MP, 5mg/kg, i.p.) administered for 10-days favors aversive memory persistence in adult rats, without any effect on the exploring behavior, locomotor activity, anxiety levels and pain perception. Enhanced performance on the inhibitory avoidance task was correlated with long-term potentiation (LTP), a phenomenon that was strengthen in hippocampal slices of rats injected with MP (5mg/kg) during 10days. Additionally, in vitro incubation with MP (30-300µM) concentration-dependently increased intracellular [Ca2+]i in cultured hippocampal neurons depolarized by KCl (35mM). In conclusion, a low daily dose of MP for 10days may promote aversive memory persistence in rats.

Intrahippocampal infusion of crotamine isolated from Crotalus durissus terrificus alters plasma and brain biochemical parameters.

Crotamine is one of the main constituents of the venom of the South American rattlesnake Crotalus durissus terrificus. Here we sought to investigate the inflammatory and toxicological effects induced by the intrahippocampal administration of crotamine isolated from Crotalus whole venom. Adult rats received an intrahippocampal infusion of crotamine or vehicle and were euthanized 24 h or 21 days after infusion. Plasma and brain tissue were collected for biochemical analysis. Complete blood count, creatinine, urea, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), creatine-kinase (CK), creatine kinase-muscle B (CK-MB) and oxidative parameters (assessed by DNA damage and micronucleus frequency in leukocytes, lipid peroxidation and protein carbonyls in plasma and brain) were quantified. Unpaired and paired t-tests were used for comparisons between saline and crotamine groups, and within groups (24 h vs. 21 days), respectively. After 24 h crotamine infusion promoted an increase of urea, GOT, GPT, CK, and platelets values (p ≤ 0.01), while red blood cells, hematocrit and leukocytes values decreased (p ≤ 0.01). Additionally, 21 days after infusion crotamine group showed increased creatinine, leukocytes, TBARS (plasma and brain), carbonyl (plasma and brain) and micronucleus compared to the saline-group (p ≤ 0.01). Our findings show that crotamine infusion alter hematological parameters and cardiac markers, as well as oxidative parameters, not only in the brain, but also in the blood, indicating a systemic pro-inflammatory and toxicological activity. A further scientific attempt in terms of preserving the beneficial activity over toxicity is required.

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad.

BACKGROUND: Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity. METHODS: The neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording - in response to indirect stimulation - and for electrophysiological measurements. RESULTS: Venom extract (50 µg/mL) increased the muscle twitch tension in PND preparations but did not significantly alter the resting membrane potential values. Electrophysiological evaluations showed that the extract (50 µg/mL) significantly augmented the frequency of miniature end-plate potential (from 38 ± 3.5 to 88 ± 15 after 60 minutes; n = 5; p < 0.05) and quantal content (from 128 ± 13 to 272 ± 34 after five minutes; n = 5; p < 0.05). Pretreatment with ouabain (1 µg/mL) for five minutes prevented the increase in quantal content (117 ± 18 and 154 ± 33 after five and 60 minutes, respectively). CONCLUSION: These results indicate that the methanolic extract of R. schneideri venom acts primarily presynaptically to enhance neurotransmitter release in mouse phrenic-diaphragm preparations.

An isoflavone from Dipteryx alata Vogel is active against the in vitro neuromuscular paralysis of Bothrops jararacussu snake venom and bothropstoxin I, and prevents venom-induced myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 µg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 µg/mL) caused irreversible paralysis. Preincubation of TM (200 µg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

The intrahippocampal infusion of crotamine from Crotalus durissus terrificus venom enhances memory persistence in rats.

Previous research has shown that crotamine, a toxin isolated from the venom of Crotalus durissus terrificus, induces the release of acetylcholine and dopamine in the central nervous system of rats. Particularly, these neurotransmitters are important modulators of memory processes. Therefore, in this study we investigated the effects of crotamine infusion on persistence of memory in rats. We verified that the intrahippocampal infusion of crotamine (1 µg/µl; 1 µl/side) improved the persistence of object recognition and aversive memory. By other side, the intrahippocampal infusion of the toxin did not alter locomotor and exploratory activities, anxiety or pain threshold. These results demonstrate a future prospect of using crotamine as potential pharmacological tool to treat diseases involving memory impairment, although it is still necessary more researches to better elucidate the crotamine effects on hippocampus and memory.

Structure-function studies on jaburetox, a recombinant insecticidal peptide derived from jack bean (Canavalia ensiformis) urease.

BACKGROUND: Ureases are metalloenzymes involved in defense mechanisms in plants. The insecticidal activity of Canavalia ensiformis (jack bean) ureases relies partially on an internal 10kDa peptide generated by enzymatic hydrolysis of the protein within susceptible insects. A recombinant version of this peptide, jaburetox, exhibits insecticidal, antifungal and membrane-disruptive properties. Molecular modeling of jaburetox revealed a prominent ß-hairpin motif consistent with either neurotoxicity or pore formation. METHODS: Aiming to identify structural motifs involved in its effects, mutated versions of jaburetox were built: 1) a peptide lacking the ß-hairpin motif (residues 61-74), JbtxΔ-ß; 2) a peptide corresponding the N-terminal half (residues 1-44), Jbtx N-ter, and 3) a peptide corresponding the C-terminal half (residues 45-93), Jbtx C-ter. RESULTS: 1) JbtxΔ-ß disrupts liposomes, and exhibited entomotoxic effects similar to the whole peptide, suggesting that the ß-hairpin motif is not a determinant of these biological activities; 2) both Jbtx C-ter and Jbtx N-ter disrupted liposomes, the C-terminal peptide being the most active; and 3) while Jbtx N-ter persisted to be biologically active, Jbtx C-ter was less active when tested on different insect preparations. Molecular modeling and dynamics were applied to the urease-derived peptides to complement the structure-function analysis. MAJOR CONCLUSIONS: The N-terminal portion of the Jbtx carries the most important entomotoxic domain which is fully active in the absence of the ß-hairpin motif. Although the ß-hairpin contributes to some extent, probably by interaction with insect membranes, it is not essential for the entomotoxic properties of Jbtx. GENERAL SIGNIFICANCE: Jbtx represents a new type of insecticidal and membrane-active peptide.

Effects of a cyanobacterial extract containing-anatoxin-a(s) on the cardiac rhythm of Leurolestes circunvagans

This work presents the effects of an anatoxin-a(s)-containing extract on a cockroach semi-isolated heart preparation and the results supporting the extract’s biological activity on acetylcholinesterase (purified from ell). The presence of the toxin in cyanobacterial strains Anabaena spiroides (ITEP-024, ITEP-025 and ITEP-026) isolated from the Tapacurá reservoir in Pernambuco, Brazil, was confirmed by means of liquid chromatography coupled to an ion-trap mass spectrometer. The anticholinesterase activity was assessed biochemically by the Ellman test and was confirmed by measuring the cockroach’s heart rate. The concentration of the extract containing the tested anatoxin-a(s) (antx-a(s)) (10, 16 and 100 μg.μL-1) inhibited the eel acetylcholinesterase (AChE) by more than 90%. The cockroach cardiac frequency increased by a maximum of about 20% within 29 min after the addition of 2.5x10³ μg of extract containing antxa (s).g-1 bw (n=9, p<0.05). Our results strongly indicate that antx-a(s) is capable of exerting biological effects on cockroach, indicating that more research might be conducted to determine its role in the environment, especially on insects.

Presynaptic effect of a methanolic extract of toad (Rhinella schneideri) poison in avian neuromuscular preparation.

The neurotoxicity of a methanolic extract of toad (Rhinella schneideri) poison was examined in chick biventer cervicis preparations. The methanolic extract (1, 3, 10 and 30µg/ml) caused concentration-dependent blockade at the three highest concentrations (time for 50% blockade, mean±SEM: 84±10, 51±3 and 12±0.8min for 3, 10 and 30µg/ml, respectively; n=6-8 each) that was preceded by significant, transient facilitation at 10µg/ml. Contractures to exogenous ACh (110µM) or KCl (20mM) were unaffected by the blockade. In curarized (d-Tc, 1µg/ml) preparations, the extract (10µg/ml) caused complete, irreversible blockade that persisted after extensive washing. The extract did not significantly alter the creatine kinase release or morphology of biventer cervicis muscle. These results indicate that the methanolic extract of R. schneideri poison acts primarily presynaptically to enhance neurotransmitter release in this avian preparation.

Implementation of the three Rs in the human hazard assessment of Brazilian medicinal plants: an evaluation of the cytotoxic and genotoxic potentials of Dipteryx alata Vogel.

In Brazil, medicinal plants are widely used by the indigenous people, which leads to a constant requirement for toxicity tests to be performed on the plant extracts. Although the current Brazilian Directive 90/2004 on the preclinical toxicity testing of phytotherapeutics recommends only in vivo tests, some Brazilian researchers would like to change this situation by implementing the Three Rs in the toxicological testing of medicinal plants. The present study evaluated the cytotoxic and genotoxic potentials of bark extracts from Dipteryx alata Vogel, a medicinal plant of the Brazilian cerrado, by using CHO-K1 (Chinese hamster ovary) cells. An IC50 value was obtained, which corresponded to 0.16mg/ml of plant extract, and from this the equivalent LD50 was determined as 705mg/kg. In order to determine the genotoxic potential of the sample, the frequency of micronucleus formation was assessed. CHO-K1 cells were exposed, during targeted mitosis, to different concentrations of plant extract and cytochalasin B, in the presence and absence of an appropriate metabolic activation system (an S9 mix). The results obtained indicated that it might be possible to implement the Three Rs in assessing the potential human hazard of medicinal plants. The publication of such data can increase awareness of the Three Rs by showing how to optimise the management of animal use, if in vivo toxicological experiments are required.

Neuromuscular and phospholipase activities of venoms from three subspecies of Bothrops neuwiedi (B. n. goyazensis, B. n. paranaensis and B. n. diporus).

The Bothrops neuwiedi (Neuwied's lancehead) species complex consists of a variety of subspecies with a wide distribution in South America. In this work, we compared the neuromuscular blockade caused by venoms from three subspecies (B. n. goyazensis, B. n. paranaensis and B. n. diporus) of this complex using chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations and investigated their phospholipase A2 (PLA2) activities and electrophoretic profiles. The order of potency of PLA2 activity was B. n. diporus>B. n. paranaensis>B. n. goyazensis. In BC preparations, B. n. goyazensis venom (50 microg/mL) was significantly (p<0.05) more active than B. n. paranaensis and B. n. diporus venoms, which did not produce a significant blockade at this time interval; after 120 min, B. n. goyazensis, B. n. paranaensis and B. n. diporus venoms (100 microg/mL) produced blockades of 57.4+/-5%, 30+/-3% and 17.4+/-7% (n=3-6 each), respectively. The three venoms inhibited contractures in response to ACh, indicating interference with postsynaptic neurotransmission. Only B. n. goyazensis and B. n. paranaensis venoms caused a long-lasting, concentration-dependent muscle contracture prior to blockade. In PND preparations, all of the venoms blocked the twitch-tension responses within 45-100 min, indicating that these preparations were more sensitive than avian preparations. There was a correlation between PLA2 activity and the time for 50% blockade in PND but not in BC preparations. SDS-PAGE showed quantitative rather than qualitative differences among the venoms. These results indicate that the venoms of the three subspecies had similar profiles of neuromuscular activity, although the relationship with PLA2 activity varied with the preparation used.

Neuromuscular and phospholipase activities of venoms from three subspeciesof Bothrops neuwiedi (B. n. goyazensis, B. n. paranaensis and B. n. diporus)

The Bothrops neuwiedi (Neuwied's lancehead) species complex consists of a variety of subspecies with a wide distribution in South America. Inthis work, we compared the neuromuscular blockade caused by venoms from three subspecies (B. n. goyazensis, B. n. paranaensis and B. n. diporus)of this complex using chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations and investigated their phospholipaseA2 (PLA2) activities and electrophoretic profiles. The order of potency of PLA2 activity was B. n. diporusNB. n. paranaensisNB. n. goyazensis. InBC preparations, B. n. goyazensis venom (50 ìg/mL) was significantly (pb0.05) more active than B. n. paranaensis and B. n. diporus venoms,which did not produce a significant blockade at this time interval; after 120 min, B. n. goyazensis, B. n. paranaensis and B. n. diporus venoms(100 ìg/mL) produced blockades of 57.4±5%, 30±3% and 17.4±7% (n=3–6 each), respectively. The three venoms inhibited contractures inresponse to ACh, indicating interference with postsynaptic neurotransmission. Only B. n. goyazensis and B. n. paranaensis venoms caused a longlasting,concentration-dependent muscle contracture prior to blockade. In PND preparations, all of the venoms blocked the twitch-tension responseswithin 45–100 min, indicating that these preparations were more sensitive than avian preparations. There was a correlation between PLA2 activityand the time for 50% blockade in PND but not in BC preparations. SDS-PAGE showed quantitative rather than qualitative differences among thevenoms. These results indicate that the venoms of the three subspecies had similar profiles of neuromuscular activity, although the relationship withPLA2 activity varied with the preparation used.

Effects of manganese ions in the neuromuscular junction

The neurotoxic effects of manganese occured mainly from its inhalation are well described in the literature. In this study we have been demonstrated the potent and reversible effect of manganese ions (Mn²+) on neuromuscular transmission using conventional myographic. The Mn²+ mechanism wich causes reversible blockade at neuromuscular junction was investigated in isolated mouse phrenic nerve-diaphragm (PND) preparations. As much Mn²+ 0.9 mM as Mn²+ 1.8 mM produced rapid neuromuscular blockade (50% in < 4 min), but only the lower concentration reversed spontaneously. The use of d-tubocurarine (5.8 µM), 3,4-diaminopyridine 3,4-DAP, 0.09 mM) and dantrolene (10 µM) excluded the involvment of nicotinic receptors, K+ channels and ryanodine receptors, respectively, as the potential target for this ion. Manganese acts an early competitive antagonist and after as an agonist of Ca²+, indicating that this ion may probably act via Ca²+ cannels...