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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 °C bath temperature, Ca 2+ - Sr 2+ 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 Ca 2+ or Sr 2+ respectively) at 24 °C or 37 °C (as controls) and in addition of B. fonsecai venom (100 μg/mL) alone or after its incubation with buffer (24 h, 23 °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 °C or 37 °C, Ca 2+ - Sr 2+ replacement, absence or presence p-BPB) were also assessed. The B. fonsecai venom caused total neuromuscular blockade after 100 min of incubation, in Ca 2+ Tyrode solution at 37 °C (usual conditions); on Sr 2+ Tyrode solution (37 °C) the twitch height were 31.7 ± 7.4% of basal, and at 24 °C (Ca 2+ 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 Ca 2+ Tyrode solution at 37 °C. The protocols delayed the time for 50% blockade: 105.7 ± 7.1 min (at 24 °C, in Ca 2+ Tyrode solution) and 71.1 ± 9.0 min (at 37 °C, in Sr 2+ 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 °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; Ca 2+ - Sr 2+ 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Guanidine has been used with some success to treat myasthenia gravis and myasthenic syndrome because it increases acetylcholine release at nerve terminals through K⁺, Na⁺ and Ca²⁺ channels-involving mechanisms. Currently, guanidine derivatives have been proposed for treatment of several diseases. Studies aimed at providing new insights to the drug are relevant. Experimentally, guanidine (10 mM) induces on mouse phrenic nerve-diaphragm (PND) preparations neurotransmission facilitation followed by blockade and a greatest secondary facilitation after its removal from bath. Herein, we hypothesized that this peculiar triphasic response may differ in muscles with distinct twitch/metabolic characteristics. Morphological alterations and contractile response of PND, extensor digitorum longus (EDL) and soleus (SOL) preparations incubated with guanidine (10 mM) for 15, 30, 60 min were analyzed. Guanidine concentrations of 5 mM (for PND and EDL) and 1 mM (for EDL) were also tested. Guanidine triphasic effect was only observed on PND regardless the concentration. The morphological alterations in muscle tissue varied along time but did not impede the PND post-wash facilitation. Higher doses (20-25 mM) did not increase EDL or SOL neurotransmission. The data suggest a complex mechanism likely dependent on the metabolic/contractile muscle phenotype; muscle fiber types and density/type of ion channels, sarcoplasmic reticulum and mitochondria organization may have profound impact on the levels and isoform expression pattern of Ca²⁺ regulatory membrane proteins so reflecting regulation of calcium handling and contractile response in different types of muscle.

Lachesis genus is one of the less studied among others from Viperidae's genera, mainly due to difficulties in obtaining the venom. Accidents by Lachesis snakes cause severe envenoming syndrome, eventually leading victims to shock. This work is part of a comprehensive study aimed at studying the venom and its effects. Herein the neurotoxicity and myotoxicity of L. muta muta venom were investigated on mouse phrenic nerve-diaphragm (PNDp) and chick biventer cervicis (BCp) preparations. For both preparations the time required to venom produces 50% neuromuscular blockade was indirectly concentration-dependent, being for PNDp: 117.6+/-6.5 min (20 microg/ml), 70.1+/-8.6 min (50 microg/ml) and 43.6+/-3.8 min (100 microg/ml), and for BCp: 28+/-1.8 min (50 microg/ml), 30.4+/-2.3 min (10 microg/ml), 50.4+/-4.3 min (5 microg/ml) and 75.2+/-0.7 min (2 microg/ml), (n=5/dose). In BCp, a venom dose of 50 microg/ml significantly reduced contractures elicited by exogenous acetylcholine (55 microM) and KCl (20 mM), as well as increased the release of creatine kinase (442.7+/-39.8 IU/l in controls vs 4322.6+/-395.2 IU/l, after 120 min of venom incubation (P<0.05). Quantification of myonecrosis in BCp indicated the doses 50 and 10 microg/ml as significantly myotoxic affecting 59.7+/-6.2%, and 20.8+/-1.2% of fibers, respectively, whereas 5 and 2 microg/ml that affected 13.5+/-0.8% and 5.4+/-0.6% of fibers, were considered weakly- and non-myotoxic, respectively. We concluded that there are neurotoxins present in the venom, the concentration of which governs its pre- (if low) or postsynaptic (if high) activity. Since myotoxicity in the avian preparation is negligible at lower venom doses, but not neurotoxicity, we suggest that this effect may contribute minimum to the venom neurotoxic effect. The BCp is more sensible than PNDp to Lachesis m. muta venom.

Whereas the presynaptic action of Crotalus durissus terrificus venom is well-established, Bothrops venoms have historically been considered to have only postsynaptic and muscular effects. However, some studies have also suggested a presynaptic action for these venoms. In this work, we used chick biventer cervicis preparations to compare the presynaptic actions of two Bothrops venoms (B. insularis and B. neuwiedi) with that of C. d. terrificus venom. At 10 microg/ml, all venoms produced irreversible blockade of the twitch tension responses, with no reduction in acetylcholine (ACh)-induced contractures and only a slight decrease in potassium induced-contractures. The times (in min) required to produce 50% neuromuscular blockade (C. d. terrificus: 16.3+/-0.7, n = 8; B. insularis: 30.0+/-1.9, n = 5; B. neuwiedi: 42.0+/-2.0, n = 8; mean +/- SEM) were significantly different among the venoms (p < 0.01). Lowering the temperature at which the experiments were done (from 37 to 24 degrees C) prevented neuromuscular blockade by the three venoms, indicating that enzyme activity may be involved in this response. At concentrations capable of causing complete neuromuscular blockade, creatine kinase release remained close to levels seen in control preparations incubated with Krebs solution alone (500-1200 IU/l). Commercial crotalic antivenom, but not bothropic antivenom, protected against the neuromuscular blockade caused by B. insularis and B. neuwiedi venoms. These observations indicate that bothropic venoms may contain components which act presynaptically in a manner similar to C. d. terrificus venom, and that at low venom concentrations a direct action on skeletal muscle does not contribute to this presynaptic neurotoxicity.

Crotoxin from Crotalus durissus cascavella venom was purified by a combination of molecular exclusion chromatography (Superdex 75 column) and HPLC molecular exclusion (Protein Pack 300SW column). Neurotoxic and myotoxic effects from C. durissus cascavella whole venom and its main fraction, the crotoxin-like, were studied in the chick biventer cervicis (CBC) nerve-muscle preparation. Both venom and its crotoxin showed significant (p < 0.05) blockade of neuromuscular transmission at concentrations as low as 0.2-1, 5 and 25 microg/ml, but no significant effect has been shown with a concentration of 0.04 microg/ml (n = 5 each). The time required to produce 50% neuromuscular blockade with the venom and its crotoxin was 53.6+/-8.2 and 65.9+/-4.9 min (0.2 microg/ml), 29.7+/-1.9 and 34.3+/-1.9 min (1 microg/ml), 24.8+/-1.6 and 21.1+/-1.5 min (5 microg/ml), 20.9+/-3.7 and 20.1+/-1.4 min (25 microg/ml), respectively. The addition to the incubation bath of acetylcholine (55 and 110 microM) or KCl (20.1 mM), either before or after the venom or the crotoxin induced contracture in the presence of a total blockade, in all the concentrations used. Morphological analysis showed that the damage caused by C. durissus cascavella venom is stronger than that caused by crotoxin. The myonecrotic picture was more marked at higher venom and crotoxin doses (1, 5 or 25 microg/ml). Only at 25 microg/ml concentrations of the venom and crotoxin, marked muscle fiber changes were detected. We concluded that the crotoxin-like and the whole venom from C. durissus cascavella possess a preponderant and quite potent neurotoxic action in this preparation, and a myotoxic action which is observed only at higher doses.

The neuromuscular effects of Bothrops neuwiedii pauloensis (jararaca-pintada) venom were studied on isolated chick biventer cervicis nerve-muscle preparations. Venom concentrations of 5-50 micro g/ml produced an initial inhibition and a secondary increase of indirectly evoked twitches followed by a progressive concentration-dependent and irreversible neuromuscular blockade. At venom concentrations of 1-20 micro g/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 micro M acetylcholine alone and cumulative concentrations of 1 micro M to 10 mM were unaffected. At venom concentrations higher than 50 micro g/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24 degrees C, the venom (50 g/ml) produced only partial neuromuscular blockade (30.7 +/- 8.0%, N = 3) after 120 min and the initial inhibition and the secondary increase of the twitch responses caused by the venom were prolonged and pronounced and the response to KCl was unchanged. These results indicate that B.n. pauloensis venom is neurotoxic, acting primarily at presynaptic sites, and that enzyme activity may be involved in this pharmacological action.

Bothropstoxin-I (BthTX-I), the principal myotoxin of Bothrops jararacussu venom, is devoid of phospholipase A(2) (PLA(2)) activity but capable of blocking neuromuscular transmission in mouse nerve-muscle preparations. In this study, the ability of crotoxin antiserum and heparin in preventing the neurotoxic and myotoxic effects of BthTX-I was investigated. Phrenic nerve-diaphragm preparations (PND) stimulated indirectly with supramaximal stimuli (0.2 ms, 0.1 Hz) were incubated with BthTX-I (20 microg/ml) alone or with BthTX-I preincubated with antiserum or heparin for 30 min at 37 degrees C prior to testing. Control preparations were incubated with Tyrode solution, antiserum or heparin alone. BthTX-I (20 microg/ml) produced 50% neuromuscular blockade in the PND preparations in 31+/-4min, with complete blockade occurring in 120 min. The antiserum and heparin significantly prevented the neuromuscular blockade caused by BthTX-I (84 +/- 4% and 100% protection, respectively). Light microscopy examination of the muscles at the end of the 120 min incubation showed that BthTX-I damaged 48 +/- 6% of the fibers. Preincubating the toxin with antivenom significantly reduced the extent of this damage (only 15 +/- 4% of fibers affected, corresponding to 69% protection, P<0.01) whereas heparin offered no protection (34 +/- 7% of fibers affected, not significantly different from that seen with toxin alone). These results show that the antivenom was more effective in neutralizing the myotoxic effects of BthTX-I than was heparin.

Bothrops jararacussu venom and its major toxin, bothropstoxin-I (BthTX-I), possess myotoxic and neurotoxic activities. The ability of commercial equine antivenom to neutralize these activities was studied in mouse isolated phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations by indirect stimulation (0.1 HZ, 0.2 ms). The time required to produce 50% neuromuscular blockade in the PND and EDL preparations was, respectively, 70 +/- 11.5 min and 58 +/- 8 min for B. jararacussu venom (50 microg/mL), and 31 +/- 6 min and 30 +/- 3 min for BthTX-I (20 microg/mL). After a 120-min incubation, the creatine kinase (CK) concentrations in the EDL preparations were 3464 +/- 346 U/L and 3422 +/- 135 U/L following exposure to venom (50 microg/mL) and BthTX-I (20 microg/mL), respectively. Antivenom neutralized the neuromuscular blockade induced by the venom and toxin in PND preparations in a dose-dependent fashion, but only partially neutralized this effect in EDL. Antivenom also effectively prevented the venom- and toxin-induced release of CK from EDL. In contrast, histological analysis showed that the morphological damage caused by B. jararacussu venom and BthTX-I in the EDL was only partially prevented by the anti- venom. These results indicate that commercial equine antiserum fully protects against the neurotoxic action of B. jararacussu and BthTX-I in PND preparations, but only partially protects against the neurotoxic and myotoxic actions of the venom and its toxin in EDL preparations. Care must therefore be exercized in extrapolating results from different preparations even when similar pharmacological or physiological responses are involved., (Copyright 2000 John Wiley & Sons, Inc.)

The effects of Bothrops pirajai snake venom on the mouse extensor digitorum longus (EDL) preparation were examined using myographic, histopathological and biochemical approaches. B. pirajai venom (10, 25 or 50 microg/ml) dose dependently and irreversibly blocked the contractile response of indirectly stimulated EDL muscle. Histopathological analysis of EDL muscle incubated with venom showed dose-dependent damage with a loss of the normal tissue structure and the appearance of highly dark, edematous fibers together with myofibrils in various stages of condensation. At high doses of venom (50 microg/ml), loss of muscle cells was observed. In non-stimulated EDL, B. pirajai venom (10 and 50 microg/ml) caused a time-dependent release of CK which was maximal after 120 min. These results suggest that a component(s) present in the B. pirajai venom has a direct myolytic action on the skeletal muscle.

The effect of Philodryas olfersii Duvernoy's secretion was studied in vivo in mice and chicks as well as in the mouse phrenic nerve-diaphragm and the chick biventer cervicis preparations. The whole secretion (20-40 micrograms/ml) increased the creatine kinase (CK) levels in mice but had no effect on the mouse phrenic nerve-diaphragm preparation. In the chick, the secretion caused head drop and paresia as well as irreversible blockade of the twitch-tension evoked by indirect stimulation in the chick biventer cervicis preparation (50% paralysis in 34.5 +/- 2.7 min, n = 4). The secretion also caused muscle contracture (30% of the maximal twitch-tension generated) after a latency of nearly 9 min. Following fractionation on a Superose 12 FPLC column, the neuromuscular activity was recovered in the high mol. wt fraction (Peak I). At a concentration of 10 micrograms/ml in the chick biventer cervicis preparation, Peak I caused 50% paralysis within 18.5 +/- 3.0 min (n = 4), and evoked a strong contracture (70% of the maximal twitch-tension generated). The contractile responses of the chick preparation to ACh and KCL were partially blocked (90%) by the whole secretion and totally blocked by Peak I. CK release was increased by the whole secretion but not by Peak I. The whole secretion also produced various degrees of muscle cell lysis and extensive widening of the intercellular spaces. The latter showed a loosely arranged membranous network. In general, Peak I caused only minor morphological alterations compared with the whole secretion, although these were still significantly different from those observed in the control preparations. The changes principally involved hypercontraction of the muscle fibers. Based on the above results, we conclude that Peak I contains the factor(s) responsible for the in vitro effects on neuromuscular transmission, whereas the direct myotoxic effect is apparently caused by at least one other component of the Duvernoy's secretion.