Neurotoxicity of Micrurus lemniscatus lemniscatus (South American coralsnake) venom in vertebrate neuromuscular preparations in vitro and neutralization by antivenom

We investigated the effect of South American coralsnake (Micrurus lemniscatus lemniscatus) venom on neurotransmission in vertebrate nerve-muscle preparations in vitro. The venom (0.1-30 µg/ml) showed calcium-dependent PLA2 activity and caused irreversible neuromuscular blockade in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, contractures to exogenous acetylcholine and carbachol (CCh), but not KCl, were abolished by venom concentrations ≥ 0.3 µg/ml; in PND preparations, the amplitude of the tetanic response was progressively attenuated, but with little tetanic fade. In low Ca2+ physiological solution, venom (10 µg/ml) caused neuromuscular blockade in PND preparations within ~ 10 min that was reversible by washing; the addition of Ca2+ immediately after the blockade temporarily restored the twitch responses, but did not prevent the progression to irreversible blockade. Venom (10 µg/ml) did not depolarize diaphragm muscle, prevent depolarization by CCh, or cause muscle contracture or histological damage. Venom (3 µg/ml) had a biphasic effect on the frequency of miniature end-plate potentials, but did not affect their amplitude; there was a progressive decrease in the amplitude of evoked end-plate potentials. The amplitude of compound action potentials in mouse sciatic nerve was unaffected by venom (10 µg/ml). Pre-incubation of venom with coralsnake antivenom (Instituto Butantan) at the recommended antivenom:venom ratio did not neutralize the neuromuscular blockade in PND preparations, but total neutralization was achieved with a tenfold greater volume of antivenom. The addition of antivenom after 50% and 80% blockade restored the twitch responses. These results show that M. lemniscatus lemniscatus venom causes potent, irreversible neuromuscular blockade, without myonecrosis. This blockade is apparently mediated by pre- and postsynaptic neurotoxins and can be reversed by coralsnake antivenom

Role of phospholipases A2 to in vitro neuromuscular and myotoxic activities of Bothrops fonsecai venom

Orientador: Léa Rodrigues SimioniDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Neste trabalho, investigou-se a participação da atividade PLA2 nas ações do veneno de B. fonsecai sobre preparações neuromusculares in vitro: extensor digitorum longus de camundongo (EDL), nervo frênico-diafragma de camundongo (NFD) e biventer cervicis de pintainho (BCp). A atividade PLA2 do veneno total foi inibida usando-se diferentes métodos como: diminuição da temperatura, substituição de cálcio por estrôncio da solução nutritiva e modificação química das PLA2 com bromofenacil brometo (p-BPB). A partir do veneno total, uma fração PLA2 foi isolada e suas atividades neuromusculares e miotóxica caracterizadas. Observou-se que o veneno de B. fonsecai (100 ?/ml) é provido de ações miotóxica (24,5 ± 1,86 % de lesão celular) e bloqueadora da junção neuromuscular de preparações EDL (bloqueio total em 100 minutos de incubação). A medida da atividade fosfolipásica mostrou diferentes graus de inibição (5,26 ± 0,08 nmol HCl, 1,27 ± 0,36 nmol HCl e inibição total para diminuição da temperatura, substituição de cálcio por estrôncio e modificação química, respectivamente) quando comparada à atividade do veneno total em condições normais (8,50 ± 0,51 nmol HCl). O veneno apresentou ainda atividades proteolítica (109,71 ± 5,87 UP/?g) e amidolítica (4238,00 ± 137,48 nmol/min), que foram reduzidas pela diminuição da temperatura (46,69 ± 1,51 UP/?g e 3112,26 ± 31,04 nmol/min), mas não sofreram inibição após a substituição de cálcio por estrôncio (214,61 ± 1,44 UP/?g e 3927,73 ± 31,04 nmol/min). A modificação química inibiu significativamente as atividades proteolítica (6,90 ± 7,01 UP/?g) e amidolítica (381,33 ± 49,78 nmol/min). Observou-se uma correlação positiva entre a inibição da atividade PLA2 e o grau de lesão celular (20,59 ± 1,61, 9,86 ± 0,76 e 3,75 ± 0,54 % de células lesadas após a diminuição da temperatura, a substituição de cálcio por estrôncio e modificação química, respectivamente), porém houve menor redução no bloqueio neuromuscular (46,43 ± 6,98, 68,33 ± 7,40 e 11,32 ± 5,67 % em 100 minutos de incubação após a diminuição da temperatura, substituição de cálcio por estrôncio e a modificação química, respectivamente). A fração PLA2 isolada apresentou características típicas das PLA2 Asp49, ou seja, cataliticamente ativas. Em preparações EDL (30 ?g/ml) causou bloqueio neuromuscular (33,4± 4,1 % após 120 minutos de incubação) e miotoxicidade (27,6 ± 1,6 % de células lesadas). O bloqueio neuromuscular foi menor em preparações BCp (8,2 ± 1,7 %), mas maior em NFD (55,8 ± 5,6 %), sem apresentar variação significativa na resposta miotóxica (20,16 ± 2,69 e 20,33 ± 2,37 % em NFD e BCp, respectivamente). Em preparações NFD sob estimulação direta, o bloqueio neuromuscular foi praticamente ausente (14,8 ± 3,5). A diminuição da temperatura e a substituição de cálcio por estrôncio reduziram a miotoxicidade (11,00 ± 0,90 e 1,33 ± 0,88 %, respectivamente) e aboliu o bloqueio neuromuscular nesta preparação. Concluiu-se que a miotoxicidade não está diretamente relacionada ao bloqueio neuromuscular do veneno de B. fonsecai. A atividade PLA2 parece desempenhar importante papel na lesão tecidual, mas pouco comprometimento na transmissão neuromuscular. Outras classes de proteínas ou PLA2 Lys49 parecem responsáveis pelo bloqueio neuromuscular produzido pelo venenoAbstract: In this work, we investigated the involvement of PLA2 activity to in vitro neuromuscular actions of B. fonsecai venom on mouse extensor digitorum longus (EDL), mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC). The venom PLA2 activity was inhibited using different methods, such as reduction of temperature, substitution of calcium by strontium in the nutritive solution and chemical modification of PLA2 with bromophenacyl bromide (p-BPB). A PLA2 fraction was isolated and its neuromuscular and myotoxic activities were characterized. It was observed that the venom of B. fonsecai (100 ?g/ml) is provided of myotoxic (24.5 ± 1.86% of cell damage) and neuromuscular blocking actions in EDL (total block after 100 minutes of incubation). The measurement of phospholipase activity showed varying degrees of inhibition (5.26 ± 0.08 nmol HCl, 1.27 ± 0.36 nmol HCl and complete inhibition in lower temperature, calcium substitution and chemical modification, respectively) when compared to the venom under normal conditions (8.50 ± 0.51 nmol HCl). The venom still showed proteolytic activity (109.71 ± 5.87 PU/ug) and amidolytic (4238.00 ± 137.48 nmol/min), which were reduced by the decrease of temperature (46.69 ± 1.51 PU/ug and 3112.26 ± 31.04 nmol/min), but suffered no inhibition after calcium replacement by strontium (214.61 ± 1.44 PU/ug and 3927.73 ± 31.04 nmol/min). The chemical modification significantly inhibited the proteolytic (6.90 ± 7.01 PU/ug) and amidolytic activity (381.33 ± 49.78 nmol/min). There was a positive correlation between inhibition of PLA2 activity and the degree of cell damage (20.59 ± 1.61, 9.86 ± 0.76 and 3.75 ± 0.54% of injured cells after lowering the temperature, replacement of calcium and strontium chemical modification, respectively), but there was less reduction in neuromuscular blockade (46.43 ± 6.98, 68.33 ± 7.40 and 11.32 ± 5.67% in 100 minutes of incubation after lowering the temperature, calcium substitution and chemical modification, respectively). The isolated PLA2 fraction had typical characteristics of PLA2 Asp49 (catalytically active). In EDL preparations (30 ?g/ml) caused neuromuscular blockade (33.4 ± 4.1% after 120 minutes of incubation) and myotoxicity (27.6 ± 1.6% of injured cells). Neuromuscular blockade was lower in BC preparations (8.2 ± 1.7%), but higher in NFD (55.8 ± 5.6%). No significant change in myotoxicity was observed between the two preparations (20.16 ± 2.69 and 20.33 ± 2.37% in PND and BC, respectively). In PND under direct stimulation the neuromuscular blockade was virtually absent (14.8 ± 3.5%). Both decrease of the temperature and calcium substitution reduced the myotoxicity (11.00 ± 0.90 and 1.33 ± 0.88%, respectively), with abolition of neuromuscular blockade. It was concluded that the myotoxicity is not directly related to the neuromuscular blockade produced by B. fonsecai venom. The PLA2 activity seems to play an important role in tissue damage, but little impairment in neuromuscular transmission. Other classes of proteins or even Lys49 PLA2 seems to be responsible for the neuromuscular blockadeMestradoFarmacologiaMestre em Farmacologia158602/2013-7CNP

Influence Of Phospholipasic Inhibition On Neuromuscular Activity Of Bothrops Fonsecai Snake Venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Bothrops fonsecai (B. fonsecai), a pitviper endemic to southeastern Brazil, has a venom mainly composed by snake venom phospholipases (PLA(2)) and metalloproteases, compounds that could interfere with neuromuscular junction in vitro. In this work, we investigated the role of PLA(2) in the myotoxicity and neuromuscular blockade caused by B. fonsecai venom using different procedures frequently associated with PLA(2) activity inhibition: 24 degrees C bath temperature, Ca2+-Sr2+ replacement and chemical modification with p-bromophenacyl bromide (p-BPB). Mice extensor digitorum longus preparations (EDL) were incubated with usual or modified Tyrode solution (prepared with Ca2+-or Sr2+ respectively) at 24 degrees C or 37 degrees C (as controls) and in addition of B. fonsecai venom (100 jig/mL) alone or after its incubation with buffer (24 h, 23 degrees C) on the absence (alkylation control) and presence of p-BPB; all muscle were processed for histological analysis. The PLA(2), proteolytic and amidolytic activities under the same conditions (24 degrees C or 37 degrees C, Ca2+-Sr2+ replacement, absence or presence p-BPB) were also assessed. The B. fonsecai venom caused total neuromuscular blockade after 100 min of incubation, in Ca2+ Tyrode solution at 37 degrees C (usual conditions); on Sr2+ Tyrode solution (37 degrees C) the twitch height were 31.7 +/- 7.4% of basal, and at 24 degrees C (Ca2+ Tyrode solution) were 53.6 +/- 7.0% of basal. The alkylation of PLA(2) with p-BPB promoted a great blockade decrease at 100 min of incubation (88.7 +/- 5.7% of basal), but it was also observed on alkylation control preparations (66.2 +/- 6.6%). The venom produced 50% of blockade at 40.5 +/- 5.9 min, in Ca2+ Tyrode solution at 37 degrees C. The protocols delayed the time for 50% blockade: 105.7 +/- 7.1 min (at 24 degrees C, in Ca2+-Tyrode solution) and 71.1 +/- 9.0 min (at 37 degrees C. in Sr2+ Tyrode solution). Regarding p-BPB incubation and alkylation control preparations, 50% of blockade was not reached during the 120 min of venom incubation. Regarding to enzymatic activities, the 24 degrees C protocol reduced not only PLA(2) (to 62.3%) but also proteolytic (52.3%) and amidolytic (73.4%) activities, as well as observed on p-BPB alkylation protocol which markedly inhibited all enzymes (<10%). The alkylation control promoted the same proteolytic and amidolytic inhibition but no reduction of PLA(2) activity; Ca2+-Sr2+ replacement reduced only the PLA(2) activity (to 15.3%). We observed a strict relation between the inhibition of PLA(2) activity and the myotoxicity. On the other hand, this relation was not observed with neuromuscular blockade, suggesting that blockade and muscle damage may not be strictly related. It suggests that the neuromuscular blockade may be induced by non-catalytic PLA(2) or other venom components, such as metalloproteinases. (C) 2017 Elsevier Ltd. All rights reserved.1303543Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil) [158602/2013-7]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP, Brazil)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

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

Pharmacological Properties of Vochysia Haenkeana (Vochysiaceae) Extract to Neutralize the Neuromuscular Blockade Induced by Bothropstoxin-I (Lys49 Phospholipase A2) Myotoxin

Purpose: Bothrops snakes are responsible for more than 70 % of snakebites every year in Brazil and their venoms cause severe local and systemic damages. The pharmacological properties of medicinal plants have been widely investigated in order to discover new alternative treatments for different classes of diseases including neglected tropical diseases as envenomation by snakebites. In this work, we have investigated the ability of Vochysia haenkeana stem barks extract (VhE) to neutralize the neuromuscular effects caused by Bothropstoxin-I (BthTX-I), the major phospholipase A2 (PLA2) myotoxin from B. jararacussu venom. Methods: The biological compounds of VhE were analysed under thin layer chromatography (TLC) and its neutralizing ability against BthTX-I was assessed through twitch-tension recordings and histological analysis in mouse phrenic nerve-diaphragm (PND) preparations. The antimicrobial activity of VhE was assessed against S. aureus, E. coli and P. aeruginosa strains. The aggregation activity of VhE was analysed under protein precipitation assay. Results: VhE showed the presence of phenolic compound visualized by blue trace under TLC. VhE abolished the neuromuscular blockade caused by BthTX-I applying the pre-toxin incubation treatment and partially neutralized the BthTX-I action under post-toxin incubation treatment; VhE contributed slightly to decrease the myotoxicity induced by BthTX-I. The neutralizing mechanism of VhE may be related to protein aggregation. VhE showed no antimicrobial activity. Conclusion: V. haenkeana extract which has no antimicrobial activity exhibited neutralizing ability against the neuromuscular blockade caused by BthTX-I and also contributed to decrease its myotoxicity. Protein aggregation involving phenolic compounds may be related in these protective effects