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.

The crude venom of Bothrops jararacussu (Bjssu) is known to induce muscular paralysis in vitro. Many studies have shown that various substances, including heparin, neutralize the damage caused by snake venom. In the present study, the ability of heparin (Hep) and commercial bothropic antivenom (CBA) to neutralize neuromuscular effects of Bjssu venom, at different time-points, was analyzed. Mouse phrenic nerve-diaphragm preparation was used through a conventional myographic technique, following five different protocols: Group 1 was incubated with Bjssu (40 µg/mL) without any other treatment; Groups 2 and 3 were pretreated with heparin (1 µL/mL) and CBA (120 µL/mL), respectively, for 15 minutes before venom addition; Group 4 after 50% neuromuscular blockade induced by Bjssu crude venom received 1 µL/mL of heparin while Group 5 received a mixture of Hep:CBA:Bjssu. Control preparations (Tyrode) were treated with Hep and CBA (mean ± SEM; n = 3-6). After 120 minutes of venom incubation, Group 1 preparations presented twitch-tension of 12 ± 2%. However, in Groups 2 and 3, the neutralizations were 92 ± 1.9% and 81 ± 6%, respectively. The heparin addition, after 50% neuromuscular blockade by Bjssu, produced 40 ± 6% muscular response after 120 minutes of incubation. Hep:CBA:Bjssu mixture displayed a protective effect of 84 ± 10% against venom action. In conclusion, heparin and commercial bothropic antivenom efficiently neutralized the neurotoxic effects caused by B. jararacussu crude venom, even at different incubation time-points.

In the present study, manganese (Mn2+), a neuromuscular blocker with pre and postsynaptic actions, was used to verify the neurotoxicity and myotoxicity induced by Crotalus durissus terrificus (Cdt) and Bothrops jararacussu (Bjssu) venoms in biventer cervicis preparations (BCp). Preparations pretreated with 0.66 and 1.6mM Mn2+ did not affect Cdt venom-induced blockage nor change KCl-induced contracture but partially reduced ACh-induced contracture. However, both Mn2+ concentrations partially hindered Bjssu venom-induced blockage after washing the preparations with Krebs solution, and only 1.6mM Mn2+ preparations significantly recovered ACh-induced contracture. The effect of Cdt venom myotoxicity on contractile responses was different from that of Bjssu venom myotoxicity. Pretreatment with 1.6mM Mn2+ partially reduced muscle damage percentage and creatine kinase (CK) activity (U/l) induced by both venoms. In conclusion, Mn2+ interfered in ACh-induced contracture of the nicotinic receptor; did not prevent Cdt venom neurotoxicity but partially reduced its myotoxicity in vitro due to the stabilizing action of this venom on the sarcolemmal membrane; and partially attenuated myotoxicity and neuromuscular blockage induced by Bjssu venom. The Mn2+ dual action (pre and postsynaptic) is useful to study snake venoms since most of them present one or both of these actions; besides, Mn2+ allowed recovering coherent interpretation of experimental versus clinical results.

Two presynaptic phospholipases A2 (PLA2), neuwieditoxin-I (NeuTX-I) and neuwieditoxin-II (NeuTX-II), were isolated from the venom of Bothrops neuwiedi pauloensis (BNP). The venom was fractionated using molecular exclusion HPLC (Protein-Pak 300SW column), followed by reverse phase HPLC (µBondapak C18 column). Tricine-SDS-PAGE in the presence or absence of dithiothreitol showed that NeuTX-I and NeuTX-II had a molecular mass of approximately 14 kDa and 28kDa, respectively. At 10µg/ml, both toxins produced complete neuromuscular blockade in indirectly stimulated chick biventer cervicis isolated preparation without inhibiting the response to acetylcholine, but NeuTX-II reduced the response to KCl by 67.0±8.0% (n=3; p<0.05). NeuTX-I and NeuTX-II are probably responsible for the presynaptic neurotoxicity of BNP venom in vitro. In fact, using loose patch clamp technique for mouse phrenic nerve-diaphragm preparation, NeuTX-I produced a calcium-dependent blockade of acetylcholine release and caused appearance of giant miniature end-plate potentials (mepps), indicating a pure presynaptic action. The N-terminal sequence of NeuTX-I was DLVQFGQMILKVAGRSLPKSYGAYGCYCGWGGRGK (71% homology with bothropstoxin-II and 54% homology with caudoxin) and that of NeuTX-II was SLFEFAKMILEETKRLPFPYYGAYGCYCGWGGQGQPKDAT (92% homology with Basp-III and 62% homology with crotoxin PLA2). The fact that NeuTX-I has Q-4 (Gln-4) and both toxins have F-5 (Phe-5) and Y-28 (Tyr-28) strongly suggests that NeuTX-I and NeuTX-II are Asp49 PLA2.

Numerous plants are used as snakebite antidotes in Brazilian folk medicine, including Casearia sylvestris Swartz, popularly known as guaçatonga. In this study, we examined the action of a hydroalcoholic extract from C. sylvestris on the neuromuscular blockade caused by bothropstoxin-I (BthTX-I), a myotoxin from Bothrops jararacussu venom, in mouse isolated phrenic nerve-diaphragm (PND) preparations. Aqueous (8 and 12 mg/ml, n=4 and 5, respectively) and hydroalcoholic (12 mg/ml, n=12) extracts of the leaves of C. sylvestris caused facilitation in PND preparations followed by partial neuromuscular blockade. BthTX-I (20 µg/ml, n=4) caused 50% paralysis after 65±15 min (mean ± S.E.M). Preincubation (30 min at 37° C) of BthTX-I (20 µg/ml, n=4) with a concentration of the hydroalcoholic extract (4 mg/ml) that had no neuromuscular activity, such as the control (n=5), prevented the neuromuscular blockade caused by the toxin. This protection may be mediated by compounds such as flavonoids and phenols identified by thin-layer chromatography and colorimetric assays.

The pharmacological effects of Bothrops neuwiedi pauloensis venom on mouse phrenic nerve-diaphragm (PND) preparations were studied. Venom (20 mug/ml) irreversibly inhibited indirectly evoked twitches in PND preparations (60 ± 10% inhibition, mean ± SEM; p<0.05; n=6). At 50 mug/ml, the venom blocked indirectly and directly (curarized preparations) evoked twitches in mouse hemidiaphragms. In the absence of Ca2+, venom (50 mug/ml), produced partial blockade only after an 80 min incubation, which reached 40.3 ± 7.8% (p<0.05; n=3) after 120 min. Venom (20 mug/ml) increased (25 ± 2%, p< 0.05) the frequency of giant miniature end-plate potentials in 9 of 10 end-plates after 30 min and the number of miniature end-plate potentials which was maximum (562 ± 3%, p<0.05) after 120 min. During the same period, the resting membrane potential decreased from - 81 ± 1.4 mV to - 41.3 ± 3.6 mV 24 fibers; p<0.01; n=4) in the end-plate region and from - 77.4 ± 1.4 to -44.6 ± 3.9 mV (24 fibers; p<0.01; n=4) in regions distant from the end-plate. These results indicate that B. n. pauloensis venom acts primarily at presynaptic sites. They also suggest that enzymatic activity may be involved in this pharmacological action.

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 µg/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 µg/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 µM acetylcholine alone and cumulative concentrations of 1 µM to 10 mM were unaffected. At venom concentrations higher than 50 µg/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24ºC, the venom (50 µg/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.

Bothrops jararacussu venom and its major toxin bothropstoxin-I (BthTX-I) possess myotoxic and neurotoxic properties. The efficacy of a rabbit antivenom raised against B. jararacussu venom in the neutralization of physiological, biochemical, and morphological changes induced by the venom and its major toxin BthTX-I was studied in mouse isolated phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations. The times required for 50% neuromuscular blockade in PND and EDL preparations for venom were 70+11.5 (S.E.M., n=5) min and 58+8 (n=16) (50 mu g/mL), and for BthTX-I 31+6 (n=3) min and 30+3 (n=5) min (20 mu g/mL), respectively. After 120 min incubation, creatine kinase (CK) concentrations in solution containing the EDL preparations were 3464+346 U/L after exposure to venom (50 mu g/mL, n=5) and 3422+135 U/L to BthTX-I (20mu g/mL, n=4), respectively. Rabbit antivenom dose-dependently neutralized venom and toxin-induced neuromuscular blockade in both preparations and effectively prevented venom and toxin-induced CK release from EDL. Histological analysis showed that rabbit antivenom neutralized morphological damage caused by B. jararacussu venom and BthTX-I in EDL preparations. These results indicate that rabbit antivenom effectively neutralized the biological activities of B. jararacussu venom and BthTX-I.

Snake venoms frequently vary in composition. In this work, we compared the neurotoxic and myotoxic activities of 16 lots of Bothrops neuwiedii venoms from different regions of Brazil, using chick biventer cervicis preparations. The neuromuscular blockade varied from 2% to 100% after 120 min incubation with venoms (50 mug/ml). In all cases, this blockade was irreversible and concentration-dependent; at low concentrations (10-20 mug/ml), 15 of the 16 venom lots failed to abolish responses to acetylcholine (110 muM), but blocked responses to KCl (13.4 muM), and induced contracture. At 5-20 mug/ml, the most active venom totally blocked twitch-tension without affecting responses to acetylcholine and KCl. Polyacrylamide gel electrophoresis for basic proteins showed that the most active samples contained a band that was absent in the less active venoms. These results indicate that there may be considerable intraspecific variation in the neurotoxic activity of B. neuwiedii venoms, whereas myotoxic activity is less variable.