RESUMO
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 »-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 percent and 100 percent 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.
Assuntos
Animais , Quitosana , Nanopartículas , Venenos de EscorpiãoRESUMO
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 »-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 percent and 100 percent 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.(AU)
Assuntos
Animais , Venenos de Serpentes/isolamento & purificação , Venenos de Serpentes/farmacologia , Análise de Sequência de Proteína/métodos , Análise de Sequência de Proteína/veterinária , Cromatografia de Fase Reversa/métodos , Cromatografia de Fase Reversa/veterinária , Receptores Pré-Sinápticos/antagonistas & inibidoresRESUMO
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 percent 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 percent. However, in Groups 2 and 3, the neutralizations were 92 ± 1.9 percent and 81 ± 6 percent, respectively. The heparin addition, after 50 percent neuromuscular blockade by Bjssu, produced 40 ± 6 percent muscular response after 120 minutes of incubation. Hep:CBA:Bjssu mixture displayed a protective effect of 84 ± 10 percent 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.
Assuntos
Animais , Masculino , Antivenenos , Bothrops , Venenos de Crotalídeos , Heparina/uso terapêutico , RatosRESUMO
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 percent 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 percent. However, in Groups 2 and 3, the neutralizations were 92 ± 1.9 percent and 81 ± 6 percent, respectively. The heparin addition, after 50 percent neuromuscular blockade by Bjssu, produced 40 ± 6 percent muscular response after 120 minutes of incubation. Hep:CBA:Bjssu mixture displayed a protective effect of 84 ± 10 percent 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.(AU)
Assuntos
Animais , Masculino , Venenos de Crotalídeos , Bothrops , Heparina/uso terapêutico , Antivenenos , RatosRESUMO
We have previously isolated a Lys49 phospholipase A(2) homolog (BaTX) from Bothrops alternatus snake venom using a combination of molecular exclusion chromatography and reverse phase HPLC and shown its ability to cause neuromuscular blockade. In this work, we describe a one-step procedure for the purification of this toxin and provide further details of its neuromuscular activity. The toxin was purified by reverse phase HPLC and its purity and molecular mass were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry, amino acid analysis and N-terminal sequencing. BaTX (0.007-1.4 microM) produced time-dependent, irreversible neuromuscular blockade in isolated mouse phrenic nerve-diaphragm and chick biventer cervicis preparations (time to 50% blockade with 0.35 microM toxin: 58+/-4 and 24+/-1 min, respectively; n=3-8; mean+/-S.E.) without significantly affecting the response to direct muscle stimulation. In chick preparations, contractures to exogenous acetylcholine (55 and 110 microM) or KCl (13.4 mM) were unaltered after complete blockade by all toxin concentrations. These results, which strongly suggested a presynaptic mechanism of action for this toxin, were reinforced by (1) the inability of BaTX to interfere with the carbachol-induced depolarization of the resting membrane, (2) a significant decrease in the frequency and amplitude of miniature end-plate potentials, and (3) a significant reduction (59+/-4%, n=12) in the quantal content of the end-plate potentials after a 60 min incubation with the toxin (1.4 microM). In addition, a decrease in the organ bath temperature from 37 degrees C to 24 degrees C and/or the replacement of calcium with strontium prevented the neuromuscular blockade, indicating a temperature-dependent effect possibly mediated by enzymatic activity.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Bloqueadores Neuromusculares/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Fosfolipases A2/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Animais , Cálcio/química , Embrião de Galinha , Agonistas Colinérgicos/farmacologia , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Diafragma/efeitos dos fármacos , Diafragma/inervação , Relação Dose-Resposta a Droga , Estimulação Elétrica , Eletroforese em Gel de Poliacrilamida , Masculino , Camundongos , Potenciais Pós-Sinápticos em Miniatura , Peso Molecular , Bloqueadores Neuromusculares/química , Bloqueadores Neuromusculares/isolamento & purificação , Fosfolipases A2/química , Fosfolipases A2/isolamento & purificação , Nervo Frênico/efeitos dos fármacos , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transmissão Sináptica/efeitos dos fármacos , Temperatura , Fatores de TempoRESUMO
Toxins that block voltage-dependent K+ channels and those that modify Na+ channel gating exhibit positive inotropic effect on skeletal muscle. We compared the effect of the venom of Tityus cambridgei (Tc) and Tityus serrulatus (Ts) scorpions on mouse diaphragm force, in vitro. In indirect and direct (using D-tubocurarine 7.3 microM) stimulation, Tc, 10microg/mL, increased the contractile force, an effect prevented by tetrodotoxin (TTX) while Ts, 0.5 microg/mL, potentiated only indirectly stimulated diaphragm, thus indicating its activity is mainly mediated through acetylcholine release from nerve terminal. This effect is prevented by TTX and attenuated by the K+ channel opener cromakalim. In conclusion, our data show that while the positive inotropic effect of both venoms appears associated to the activity of Na+ and K+ channels, only Tc venom acts also directly on skeletal muscle. This finding call for further studies on Tc venom to identify the toxin responsible for its direct inotropic activity as it may have clinical applications.
Assuntos
Diafragma/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/efeitos dos fármacos , Acetilcolina/metabolismo , Animais , Cromakalim/farmacologia , Diafragma/inervação , Diafragma/metabolismo , Estimulação Elétrica , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Camundongos , Força Muscular/efeitos dos fármacos , Fármacos Neuromusculares não Despolarizantes/farmacologia , Nervo Frênico/efeitos dos fármacos , Nervo Frênico/metabolismo , Canais de Potássio/metabolismo , Canais de Sódio/metabolismo , Tetrodotoxina/farmacologia , Fatores de Tempo , Tubocurarina/farmacologiaRESUMO
Ethanolic extract of leaves of Galactia glauscescens (GGE) at concentration of 100 and 500 microg/ml prevented the neuromuscular paralysis induced by Crotalus durissus terrificus venom on mouse phrenic nerve-diaphragm preparation.
Assuntos
Venenos de Crotalídeos/toxicidade , Crotalus/fisiologia , Fabaceae/química , Extratos Vegetais/farmacologia , Animais , Diafragma/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Camundongos , Bloqueio Neuromuscular , Junção Neuromuscular/efeitos dos fármacos , Nervo Frênico/efeitos dos fármacos , Extratos Vegetais/química , Folhas de Planta/químicaRESUMO
Casearia sylvestris Sw., popularly known in Brazil as 'guaçatonga', has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C. sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects.
Assuntos
Casearia/química , Contração Muscular/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Brasil , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Diafragma/efeitos dos fármacos , Diafragma/inervação , Diafragma/fisiologia , Técnicas In Vitro , Masculino , Metanol/química , Camundongos , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/fisiologia , Extratos Vegetais/químicaRESUMO
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.
Assuntos
Animais , Venenos de Crotalídeos , Manganês/farmacologia , Manganês/uso terapêutico , Bloqueio NeuromuscularRESUMO
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 (æBondapak 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 10æg/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 percent (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 percent homology with bothropstoxin-II and 54 percent homology with caudoxin) and that of NeuTX-II was SLFEFAKMILEETKRLPFPYYGAYGCYCGWGGQGQPKDAT (92 percent homology with Basp-III and 62 percent 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.
Assuntos
Animais , Bothrops/metabolismo , Venenos de Crotalídeos , Fosfolipases A/química , Neurotoxinas/intoxicaçãoRESUMO
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.
RESUMO
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.
RESUMO
In this paper we reported the purification, the biological characterization and the amino acid sequence of two new isoforms basic 6-1 (Bj-IV) and 6-2 (Bj-V) PLA(2) D49 purified from the Bothrops jararacussu venom. The isoforms 6-1 and 6-2 had a sequence of amino acids of 121 amino acid residues 6-1: DLFEWGQMIL KETGKNPFPY YGAYGCYCGW GGRGKPKDKD TDRCCYVHDC CYKKLTGCPK TDDRYSYSWL DLTIVCGEDD PCKELCECDK AIAVCFRENL GTYNKKYRYH LKPCKKADKP C and pI value 7.83 and 6-2: DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHDC CYKKLTGCPK TDDRYSYSWL DLTIVCGEDD PCKELCECDK AIAVCFRENL GTYNKKYRYH LKPCKKADKP C with a pI value of 7.99. Skeletal muscle preparations from the young chicken have been used previously in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. Both isoforms have produced neuromuscular blockade in young chicken biventer cervicis nerve-muscle preparations in presence or absence of crotapotin crotalic (F3 and F4) indicating that catalytic activity was not essential for neuromuscular action in this preparation.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Junção Neuromuscular/efeitos dos fármacos , Fosfolipases A/química , Fosfolipases A/toxicidade , Sequência de Aminoácidos , Animais , Galinhas , Venenos de Crotalídeos/toxicidade , Crotoxina/farmacologia , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Isoenzimas/toxicidade , Dados de Sequência Molecular , Fosfolipases A/isolamento & purificação , Fosfolipases A/metabolismo , Fosfolipases A2 , Homologia de Sequência de AminoácidosRESUMO
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 mg/ml, n=4) caused 50% paralysis after 65±15 min (mean ± S.E.M). Preincubation (30 min at 37°C) of BthTX-I (20 mg/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
Assuntos
Animais , Masculino , Camundongos , Extratos Vegetais/uso terapêutico , Plantas Medicinais , Mordeduras de Serpentes , Venenos de Serpentes , Bloqueio NeuromuscularRESUMO
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 percent 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 percent (p<0.05; n=3) after 120 min. Venom (20 mug/ml) increased (25 ± 2 percent, 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 percent, 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.(AU)
Assuntos
Animais , Camundongos , Nervo Frênico , Venenos de Serpentes , Fármacos Neuromusculares , Junção Neuromuscular , Bothrops , Potenciais da MembranaRESUMO
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.
RESUMO
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.
RESUMO
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 æg/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 æg/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 æM acetylcholine alone and cumulative concentrations of 1 æM to 10 mM were unaffected. At venom concentrations higher than 50 æg/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24ºC, the venom (50 æg/ml) produced only partial neuromuscular blockade (30.7 ± 8.0 percent, 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
Assuntos
Animais , Bothrops , Venenos de Crotalídeos , Contração Muscular , Músculo Esquelético , Junção Neuromuscular , Acetilcolina , Galinhas , Cloreto de Potássio , Fatores de TempoRESUMO
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.
Assuntos
Bothrops , Venenos de Crotalídeos/intoxicação , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/inervação , Junção Neuromuscular/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Galinhas , Relação Dose-Resposta a Droga , Cloreto de Potássio/farmacologia , Fatores de TempoRESUMO
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 per cent to 100 per cent after 120 min incubation with venoms (50µg/ml). In all cases, this blockade was irreversible and concentration-dependent; at low concentrations (10-20 µg/ml), 15 of the 16 venom lots failed to abolish responses to acetylcholine (110µM), but blocked responses to KCI (13.4mM), and induced contracture. At 5-20µg/ml, the most active venom totally blocked twitch-tension without affecting responses to acetylcholine and KCI. Polyacrylamine 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. ineuwiedii venoms, whereas myotoxic activity is less variable.