RESUMEN
CONTEXT: In order to report the outcome of a patient who developed compartment syndrome after South American rattlesnake (Crotalus durissus terrificus) envenomation, confirmed by subfascial pressure measurement and magnetic resonance imaging (MRI). CASE DETAILS: A 63-year-old male was admitted 1 h after being bitten on the right elbow by a "large" snake, which was not brought for identification. Physical and laboratory features upon admission revealed two fang marks, local tense swelling, paresthesia, intense local pain, hypertension, coagulopathy, and CK = 1530 U/L (RV < 170 U/L). The case was initially treated with bothropic antivenom (80 mL, intravenously), with no improvement. Evolution within 13-14 h post-bite revealed generalized myalgia, muscle weakness, palpebral ptosis, and severe rhabdomyolysis (CK = 126,160 U/L) compatible with envenoming by C. d. terrificus. The patient was then treated with crotalic antivenom (200 mL, intravenously), fluid replacement, and urine alkalinization. Twenty-four-hour post-bite MRI showed marked muscular edema in the anterior compartment of the right forearm, with a high subfascial pressure (40 mmHg) being detected 1 h later. ELISA of a blood sample obtained upon admission, before antivenom infusion, revealed a high serum concentration of C. d. terrificus venom. No fasciotomy was performed and the patient was discharged seven days later without sequelae. CONCLUSION: Snakebite by C. d. terrificus with subfascial venom injection may lead to increased intracompartmental pressure.
Asunto(s)
Síndromes Compartimentales/etiología , Mordeduras de Serpientes/complicaciones , Animales , Antivenenos/uso terapéutico , Síndromes Compartimentales/diagnóstico , Venenos de Crotálidos/antagonistas & inhibidores , Crotalus , Antebrazo/irrigación sanguínea , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Mordeduras de Serpientes/tratamiento farmacológicoRESUMEN
CONTEXT: Rattlesnake bites in Brazil are generally caused by adult individuals, with most of the envenomed patients showing systemic manifestations that include varying degrees of neurotoxicity (acute myasthenia), rhabdomyolysis and coagulopathy, with only mild or no local manifestations. We report a case of envenoming by a juvenile South American rattlesnake (Crotalus durissus terrificus) that involved coagulopathy as the main systemic manifestation. CASE DETAILS: A 19-year-old male was admitted to our Emergency Department with coagulopathy (incoagulable PT, APTT and INR), no remarkable local manifestations and no signs/symptoms of myasthenia or rhabdomyolysis (serum CK, LDH, ALT and AST within reference levels) 5 days after being bitten by a small snake that was described as a rattlesnake but was not brought for identification at admission. The patient had already been treated in another Emergency Department with i.v. bothropic antivenom (AV) 1 h and 4 days post-bite. Based on the possibility of an unusual rattlesnake bite, crotalic AV was administered i.v., which improved the coagulation (9 h post-CroAV, INR = 2.11; 36 h post-CroAV, INR = 1.42). During hospitalization, relatives brought the snake that caused the bite, which was identified as a 38-cm long C. d. terrificus. DISCUSSION: Little is known about the clinical manifestations after bites by juvenile C. d. terrificus. This case shows that systemic envenoming by juvenile C. d. terrificus may result in coagulopathy as the main systemic manifestation, without neuromyotoxic features normally associated with bites by adult specimens. Despite the delayed administration, crotalic AV was effective in improving the blood coagulation.
Asunto(s)
Trastornos de la Coagulación Sanguínea/etiología , Mordeduras de Serpientes/complicaciones , Animales , Antivenenos/uso terapéutico , Brasil , Venenos de Crotálidos/efectos adversos , Venenos de Crotálidos/antagonistas & inhibidores , Crotalus , Humanos , Masculino , Mordeduras de Serpientes/terapia , Adulto JovenRESUMEN
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.
Asunto(s)
Bothrops , Venenos de Crotálidos/enzimología , Bloqueantes Neuromusculares/farmacología , Unión Neuromuscular/efectos de los fármacos , Fosfolipasas A2/farmacología , Terminales Presinápticos/efectos de los fármacos , Animales , Calcio/química , Embrión de Pollo , Agonistas Colinérgicos/farmacología , Cromatografía Líquida de Alta Presión , Venenos de Crotálidos/química , Venenos de Crotálidos/aislamiento & purificación , Venenos de Crotálidos/farmacología , Diafragma/efectos de los fármacos , Diafragma/inervación , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Electroforesis en Gel de Poliacrilamida , Masculino , Ratones , Potenciales Postsinápticos Miniatura , Peso Molecular , Bloqueantes Neuromusculares/química , Bloqueantes Neuromusculares/aislamiento & purificación , Fosfolipasas A2/química , Fosfolipasas A2/aislamiento & purificación , Nervio Frénico/efectos de los fármacos , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transmisión Sináptica/efectos de los fármacos , Temperatura , Factores de TiempoRESUMEN
Bothrops lanceolatus venom contains a variety of enzymatic and biological activities. The present work investigated the hemolytic activity of this venom and its phospholipase A2 (PLA2). Bothrops lanceolatus venom (6.7 µg/mL) caused indirect hemolysis of cow, horse, rat and sheep erythrocytes, with horse erythrocytes being the most sensitive; no direct hemolysis was observed. Hemolysis in sheep erythrocytes was concentration-dependent (5-11.7 µg/mL) and markedly attenuated by heating the venom for 30 minutes at ≥ 40°C and by the PLA2 inhibitor p-bromophenacyl bromide. An acidic PLA2 (5 µg/mL) purified from B. lanceolatus venom also caused hemolysis. This PLA2 showed immunoprecipitin lines with antivenom against B. lanceolatus, which suggests that the enzymatic and hemolytic activities of this enzyme may be neutralized during antivenom therapy. These results indicate that B. lanceolatus venom and its PLA2 can cause hemolysis in vitro.(AU)
Asunto(s)
Técnicas In Vitro , Bothrops lanceolatus , Venenos Elapídicos/toxicidad , Enzimas , Fosfolipasas A2 , Productos Biológicos , HemólisisRESUMEN
Venom of the South American rattlesnake, Crotalus durissus terrificus (Cdt), presents myotoxic and neurotoxic outcomes, but reports on its effects on the liver are scarce. This study examined the hepatotoxicity resulting from Cdt venom administration (100, 200 and 300 miug/kg) in male Wistar rats. Animais were studies at 3, 9 and 12 hours after venom injection. The hepatotoxicity was assessed through serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), gamma glutamyl transferase (GGT), bilirrubin and also by histopathological evaluation. All the different concentrations of Cdt venom resulted in increased levels of hepatic enzymes, when compared with the control group, except for the 100 miug/kg dose, which presented normal levels at 9 and 12 hours after venom administration. Bilirrubin levels remained unchanged by Cdt venom. Histological analysis revealed endothelial damage, inflammatory cell infiltration, as well as sinusoidal and portal congestion. Based on these observations, we may conclude that Cdt venom causes dose- and time-dependent hepatic damage in rats, characterized by elevated hepatic enzyme levels and histological alterations
Asunto(s)
Animales , Masculino , Hígado/anatomía & histología , Hígado , Venenos de Crotálidos/envenenamiento , Venenos de Crotálidos/toxicidad , Alanina Transaminasa/administración & dosificación , Aspartato Aminotransferasas/administración & dosificación , Fosfatasa Alcalina/administración & dosificación , Ratas WistarRESUMEN
Bothrops lanceolatus venom contains a variety of enzymatic and biological activities. The present work investigated the hemolytic activity of this venom and its phospholipase A2 (PLA2). Bothrops lanceolatus venom (6.7 µg/mL) caused indirect hemolysis of cow, horse, rat and sheep erythrocytes, with horse erythrocytes being the most sensitive; no direct hemolysis was observed. Hemolysis in sheep erythrocytes was concentration-dependent (5-11.7 µg/mL) and markedly attenuated by heating the venom for 30 minutes at 40°C and by the PLA2 inhibitor p-bromophenacyl bromide. An acidic PLA2 (5 µg/mL) purified from B. lanceolatus venom also caused hemolysis. This PLA2 showed immunoprecipitin lines with antivenom against B. lanceolatus, which suggests that the enzymatic and hemolytic activities of this enzyme may be neutralized during antivenom therapy. These results indicate that B. lanceolatus venom and its PLA2 can cause hemolysis in vitro.
RESUMEN
Venom of the South American rattlesnake, Crotalus durissus terrificus (Cdt), presents myotoxic and neurotoxic outcomes, but reports on its effects on the liver are scarce. This study examined the hepatotoxicity resulting from Cdt venom administration (100, 200 and 300 µg/kg) in male Wistar rats. Animals were studies at 3, 6, 9 and 12 hours after venom injection. The hepatotoxicity was assessed through serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), gamma glutamyl transferase (GGT), bilirrubin and also by histopathological evaluation. All the different concentrations of Cdt venom resulted in increased levels of hepatic enzymes, when compared with the control group, except for the 100 µg/kg dose, which presented normal levels at 9 and 12 hours after venom administration. Bilirrubin levels remained unchanged by Cdt venom. Histological analysis revealed endothelial damage, inflammatory cell infiltration, as well as sinusoidal and portal congestion. Based on these observations, we may conclude that Cdt venom causes dose- and time-dependent hepatic damage in rats, characterized by elevated hepatic enzyme levels and histological alterations.
RESUMEN
A previously healthy, 21-year-old female was admitted 5 h after being bitten in the occipital region by a pitviper presumed to be Bothrops jararaca. Physical examination revealed marked cranial and facial oedema extending to the neck and dorsum, bilateral eyelid ecchymosis, and local conjunctival and gingival bleeding. The patient was alert and complained of mild, local pain and nausea. There were no signs of neurological involvement. The main laboratory findings on admission included incoagulable blood, a platelet count of 4000/microl, and an ELISA-estimated serum venom concentration of 62.6 ng/ml. Sequential serum creatinine, urea nitrogen, sodium and potassium concentrations were normal. The case was classified as severe and, after the intravenous administration of ranitidine, chlorpheniramine and hydrocortisone, the intravenous infusion of 12 vials of undiluted bothropic equine antivenom [F(ab)(2); 10 ml/vial] was initiated. The antivenom infusion was halted after 10 vials because the patient developed a severe early reaction, although this was successfully treated with subcutaneous adrenaline and intravenous hydrocortisone. Platelet replacement (seven units) was performed and 24 h after the antivenom infusion, normal results in blood-coagulation tests and an increase in the platelet count (to 100,000/microl) were observed. No circulating venom was detected in blood samples collected 6, 12, 24 or 48 h post-admission. The patient was discharged after 4 days, with clinical improvement and no signs of local infection, and subsequent follow-up revealed no sequelae.
Asunto(s)
Antivenenos/efectos adversos , Bothrops , Venenos de Crotálidos/envenenamiento , Mordeduras de Serpientes/tratamiento farmacológico , Adulto , Animales , Femenino , Humanos , Factores Inmunológicos/uso terapéutico , Mordeduras de Serpientes/patología , Resultado del TratamientoRESUMEN
A new Phospholipase A(2) (PLA(2)) from Micrurus dumerilii carinicauda venom was isolated and its primary structure determined. This new PLA(2) showed a low enzymatic activity when compared with other PLA(2)s and it is moderately basic with an isoelectric point of 8.0. Its amino acid sequence showed the presence of 120 amino acid residues and its sequence was: NLIQFLNMIQCTTPGREPLVAFANYGCYCGRGGSGTPVDELDRCCQVHDNCYDTAKKVFGCSPYFTMYSYDCSEGKLTCKDNNTKCKAAVCNCDRTAALCFAKAPYNDKNYKIDLTKRCQ. The structural model of MIDCA1, when compared with other strong neurotoxic PLA(2)s, such as Naja naja, showed significant differences in the beta-wing and neurotoxic sites, despite the high level of amino acid sequence similarity. These observations indicate a dissociation between the biological and catalytic activity of this new PLA(2), supporting the view that other regions of the protein are involved in the biological effects.
Asunto(s)
Secuencia de Aminoácidos , Venenos Elapídicos/enzimología , Fosfolipasas A/genética , Animales , Elapidae , Modelos Moleculares , Datos de Secuencia Molecular , Fosfolipasas A/química , Fosfolipasas A/aislamiento & purificación , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
A novel protein with factor Xa-like activity was isolated from Lonomia obliqua caterpillar spicules by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The protein had a mass of 20745.7 Da, as determined by mass spectrometry, and contained four Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of the protein and the cysteine residues linked by disulfide bridges. The positions of 24 sequenced tryptic peptides, including the N-terminal, were deduced by comparison with a homologous protein from the superfamily Bombycoidea. Approximately 90% of the primary structure of the active protein was determined.
Asunto(s)
Factor Xa/aislamiento & purificación , Factor Xa/metabolismo , Lepidópteros/química , Lepidópteros/crecimiento & desarrollo , Alquilación , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Disulfuros/análisis , Disulfuros/química , Factor Xa/química , Espectrometría de Masas , Datos de Secuencia MolecularRESUMEN
A crotoxin homolog was purified from the Crotalus durissus collilineatus venom using molecular exclusion and reverse-phase HPLC. This crotoxin contained one PLA2 (Cdcolli III F6) and four crotapotin isoforms, whereas crotoxin from Crotalus durissus terrificus venom had three PLA2 iso forms and two crotapotin isoforms. SDS-PAGE showed that the C. d. collilineatus PLA2 and crotapotin had relative molecular mass of 15 and 9 kDa, respectively. Neither the PLA2 (Cdcolli III F6) nor the crotapotins (Cdcolli III F3 and F4) had any neurotoxicity in mouse phrenic nerve-diaphragm preparations when tested alone. However, when PLA2 and crotapotin were coincubated before testing, the neurotoxicity was restored to a level similar to test in the venom in native crotoxin. The two crotapotins (Cdcolli III F3 and F4) differed in their ability to inhibit PLA2 activity, perhaps because of variations in their affinities for this enzyme. Cdcolli III F6 showed allosteric enzymatic behavior, with maximal activity at pH 8.3 and 36 degrees C. Full PLA2 activity required the presence of a low Ca2+ concentration and was inhibited by Cu2+ and Zn2+ and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. These results indicate that crotoxin from C. d. collineatus venom is very similar enzymatically to crotoxin from C. d. terrificus.
Asunto(s)
Venenos de Crotálidos/enzimología , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión/métodos , Crotalus/metabolismo , Crotoxina/química , Crotoxina/aislamiento & purificación , Crotoxina/metabolismo , Crotoxina/farmacología , Electroforesis en Gel de Poliacrilamida , Femenino , Técnicas In Vitro , Concentración 50 Inhibidora , Isoenzimas/antagonistas & inhibidores , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Isoenzimas/farmacología , Cinética , Masculino , Ratones , Datos de Secuencia Molecular , Nitrobenzoatos/metabolismo , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/farmacología , Fosfolipasas A2 , Nervio Frénico/efectos de los fármacos , Homología de Secuencia de AminoácidoRESUMEN
A phosphodiesterase was purified from the venom of the snake Bothrops alternatus by a combination of gel filtration and ion exchange chromatographies. In SDS-PAGE, the enzyme gave a single band with a molecular mass of 105 kDa, which was unaltered in the presence of beta-mercaptoethanol, indicating that the protein contained no subunits. A single protein band was also observed in native PAGE. There were no contaminating 5'-nucleotidase, alkaline phosphatase and protease activities. The enzyme was recognized by commercial bothropic antiserum and gave a single band in immunoblotting. The enzyme had a pH optimum in the range of 7.5-9.5 and the optimum temperature was 60 degrees C, with activity being rapidly lost within 1 min at > or = 70 degrees C. The Km of the enzyme was 2.69 mM. PDE activity was potentiated by cobalt and, to a lesser extent, by calcium, whereas copper, manganese, zinc, EDTA, and beta-mercaptoethanol were inhibitory. These properties show that this enzyme is very similar to that isolated from other snake venoms.
Asunto(s)
Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/aislamiento & purificación , Animales , Bothrops , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cobre/farmacología , Ácido Edético/farmacología , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Cinética , Manganeso/farmacología , Mercaptoetanol/farmacología , Sefarosa/farmacología , Temperatura , Factores de Tiempo , Zinc/farmacologíaRESUMEN
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 per cent neuromuscular blockade in PND and EDL preparations for venom were 70ñ11.5 (S.E.M., n=5) min and 58ñ8 (n=16) (50 µ/mL), and for BthTX-I 31ñ6 (n=3) min and 30ñ3 (n=5) min (20 µg/mL), respectively. After 120 min incubation, creatine kinase (CK) concentrations in solution containing the EDL preparations were 3464ñ346 U/L after exposure to venom (50 µg/mL, n=5) and 3422ñ135 U/L to BthTX-I (20µg/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.
Asunto(s)
Animales , Masculino , Conejos , Ratas , Antitoxinas , Antivenenos , Venenos de Crotálidos , Conejos , BothropsRESUMEN
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.
Asunto(s)
Animales , Masculino , Bothrops , Brasil , Pollos , Miotonía , Sistema Nervioso , Neurotoxinas , Venenos de Crotálidos/efectos adversos , Venenos de Crotálidos/toxicidad , Acetilcolina , Contractura , Bloqueo NeuromuscularRESUMEN
A novel basic phospholipase A2 (PLA2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY...) showed a high degree of homology with basic D49 PLA2 myotoxins from other Bothrops venoms. Bj IV had high PLA2 activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45 degrees C. Full PLA2 activity required Ca2+ but was inhibited by Cu2+ and Zn2+, and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA2 was similar to that in the basic PLA2 of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA2 could partly explain the low PLA2 activity of Bothrops venoms.
Asunto(s)
Bothrops , Venenos de Crotálidos/química , Venenos de Crotálidos/enzimología , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/metabolismo , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Calcio/metabolismo , Cromatografía , Venenos de Crotálidos/farmacología , Crotoxina/farmacología , Técnicas In Vitro , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Isoenzimas/farmacología , Ratones , Datos de Secuencia Molecular , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/fisiología , Fosfolipasas A/química , Fosfolipasas A/farmacología , Fosfolipasas A2 , Ratas , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Antivenenos/farmacología , Venenos de Crotálidos/antagonistas & inhibidores , Crotoxina/antagonistas & inhibidores , Inmunoglobulina G/análisis , Músculo Esquelético/efectos de los fármacos , Nervio Frénico/efectos de los fármacos , Animales , Antivenenos/inmunología , Antivenenos/uso terapéutico , Bothrops , Venenos de Crotálidos/inmunología , Venenos de Crotálidos/toxicidad , Crotoxina/inmunología , Crotoxina/toxicidad , Estimulación Eléctrica , Electroforesis en Gel de Poliacrilamida , Heparina/uso terapéutico , Inmunoglobulina G/sangre , Inmunoglobulina G/aislamiento & purificación , Técnicas In Vitro , Inyecciones Subcutáneas , Masculino , Ratones , Músculo Esquelético/patología , Músculo Esquelético/fisiología , Bloqueo Neuromuscular , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/patología , Unión Neuromuscular/fisiología , Pruebas de Neutralización , Nervio Frénico/fisiología , Conejos , Factores de TiempoRESUMEN
The effects of the Brazilian herbal medicine Catuama and each of its plant constituents (Paullinia cupana, Trichilia catigua, Zingiber officinalis and Ptychopetalum olacoides) were investigated on rabbit corpus cavernosum (RbCC) using a bioassay cascade. Catuama caused short-lived and dose-dependent relaxations (11% +/- 7%, 26% +/- 5% and 82% +/- 9%, at doses of 1, 3 and 10 mg, respectively). Neither the nitric oxide synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 10 microM) nor the soluble guanylate cyclase inhibitor ODQ (10 microM) significantly affected the Catuama-induced relaxations. Similarly, the selective ATP-dependent K(+) channel (K(ATP)) blocker glibenclamide (10 microM), the muscarinic receptor antagonist atropine (1 microM) and the voltage-dependent Na(+) channel blocker tetrodotoxin (1 microM) all failed to affect significantly the Catuama-induced relaxations. These results indicate that the relaxations induced by Catuama involve neither nitric oxide release nor K(ATP) channel activation. The extracts of P. cupana, Z. officinalis and P. olacoides caused short-lived and dose-dependent RbCC relaxations, whereas T. catigua evoked long-lasting relaxations which were occasionally preceded by a brief contractile effect. The extract of P. cupana was the most active in relaxing RbCC strips. The relaxations induced by all extracts were not significantly affected by L-NAME (10 microM). The infusion of ODQ (10 microM) had no significant effect on the P. cupana- and Z. officinalis-induced relaxations but reduced by >50% (p < 0.05) those evoked by P. olacoides and T. catigua. Incubations of RbCC with Catuama(10 mg/mL for 0.25 to 5 min) caused increases of cAMP levels (143% increase at 5 min of incubation). Incubations of RbCC with P. cupana extract (1 mg/mL) increased the cAMP levels by 200% whereas higher doses (10 and 100 mg/mL) caused smaller increases in the nucleotide levels (150% and 89%, respectively). The extracts of Z. officinalis and P. olacoides (same doses) caused smaller increases of the cAMP levels compared with the P. cupana extract, whereas T. catigua (1-100 mg) did not increase the levels of this nucleotide above the basal values. Our results show that of the four extracts assayed, P. cupana was the most effective, indicating that it is the main extract responsible for the relaxing effect of Catuama on rabbit cavernosal tissue.
Asunto(s)
Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Extractos Vegetales/farmacología , Plantas Medicinales , Rosales , Estómago/efectos de los fármacos , Animales , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Extractos Vegetales/administración & dosificación , ConejosRESUMEN
Pravastatin is useful in restoring endothelium-dependent relaxation in hypercholesterolemic animals. A single intravenous bolus injection of N(omega)-nitro-L-arginine methyl ester (L-NAME), a non-specific inhibitor of NO synthase, causes myocardial necrosis and reduces coronary flow in rats. Since rats do not develop hypercholesterolemia and atherosclerosis, we have tested the hypothesis that pravastatin protects the heart from myocardial lesions induced by L-NAME in the absence of alterations in cholesterol levels and plaque formation. Male Wistar rats fed standard chow were divided into four groups: CONTROL (n=14) - rats that received tap water alone for 18 days; L-NAME (n=14) -- rats that received L-NAME (15 mg/kg, i.v.) on the 14th day of the study; PRAVASTATIN (n=11) -- rats that received pravastatin (6 mg/kg/day) in their drinking water for 18 days; PRAVASTATIN+L-NAME (n=12) -- rats that received pravastatin (6 mg/kg/day) and L-NAME (15 mg/kg, i.v.) as indicated in the preceding groups. At the end of 18 days, the rats were sacrificed and the hearts removed for stereological analysis by light microscopy. Plasma nitrate/nitrite and thromboxane B(2) concentrations were determined immediately before and after L-NAME administration. Pravastatin prevented the ischemic lesions induced by the acute inhibition of NO biosynthesis (the area of myocardial lesions in the L-NAME group was greater than in the Pravastatin+L-NAME group: 101.6 microm(2) vs. 1.2 microm(2), respectively; P<0.0001) and markedly increased the plasma nitrate/nitrate concentrations, even before L-NAME administration. There were no significant changes in the plasma thromboxane B(2) concentrations.
Asunto(s)
Anticolesterolemiantes/uso terapéutico , Cardiomiopatías/tratamiento farmacológico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Óxido Nítrico Sintasa/biosíntesis , Pravastatina/uso terapéutico , Análisis de Varianza , Animales , Cardiomiopatías/etiología , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos , Masculino , NG-Nitroarginina Metil Éster , Necrosis , Nitratos/sangre , Nitritos/sangre , Ratas , Ratas Wistar , Tromboxano B2/sangreRESUMEN
OBJECTIVE AND DESIGN: To examine the role of macrophages in the neutrophil migration induced by staphylococcal enterotoxin type A (SEA) in mice. MATERIALS AND METHODS: Peritoneal macrophages were harvested from male Swiss mice pre-treated with thioglycollate. After adhering to plastic tissue culture dishes, the cells were washed and incubated with RPMI or SEA (0.62-2.5 microg/ml) and washed again prior to further incubation with RPMI alone. The medium was then collected, sterilized and assayed for promigratory activity in the mouse peritoneal cavity. RESULTS: Mouse macrophage monolayers stimulated with SEA secreted a thermolabile neutrophil chemotactic component (MNCC-SEA) with a molecular mass >100 kDa (by ultrafiltration). This release was dose- and time-dependent and was inhibited by dexamethasone but not by indomethacin or BW755C. Dexamethasone, indomethacin, BWA4C, BW755C, BN52021, cimetidine and SR48968 had no effect on the neutrophil migration induced by MNCC-SEA while capsaicin and SR 140333 reduced this phenomenon. CONCLUSIONS: Macrophages play a key role in the neutrophil recruitment induced by SEA probably by releasing an MNCC-SEA that presumably induces neutrophil migration via a mechanism mediated by substance P.
Asunto(s)
Factores Quimiotácticos/metabolismo , Enterotoxinas/farmacología , Macrófagos Peritoneales/metabolismo , Neutrófilos/fisiología , 4,5-dihidro-1-(3-(trifluorometil)fenil)-1H-pirazol-3-amina/farmacología , Animales , Benzamidas/farmacología , Calcio/farmacología , Capsaicina/farmacología , Factores Quimiotácticos/farmacología , Quimiotaxis de Leucocito , Inhibidores de la Ciclooxigenasa/farmacología , Dexametasona/farmacología , Estabilidad de Medicamentos , Glucocorticoides/farmacología , Glucosa/farmacología , Indometacina/farmacología , Magnesio/farmacología , Masculino , Ratones , Peso Molecular , Piperidinas/farmacología , Receptores de Neuroquinina-2/antagonistas & inhibidores , Tioglicolatos/farmacologíaRESUMEN
The effect of neonatal capsaicin (8 methyl-N-vanillyl-6-nonenamide) treatment on the leucocyte infiltration into the airways and pleural cavity was investigated in rats actively sensitized with ovalbumin. The animals were neonatally injected with either capsaicin (50 mg/kg, s.c., 2nd day of life) or vehicle (10% ethanol and 10% Tween 80). At adult ages, the animals were actively sensitized with ovalbumin (200 microg, s.c.) and 14 days later they were intratracheally (or intrapleurally) challenged with ovalbumin. The substance P level in bronchoalveolar lavage fluid of the capsaicin group was reduced by >90% compared to control group (vehicle), confirming the efficacy of capsaicin treatment. In the capsaicin group, the number of neutrophils (but not of eosinophils and mononuclear cells) in bronchoalveolar lavage fluid of sensitized animals was significantly higher than the control group. Intrapleural injection of ovalbumin in sensitized rats caused a significant neutrophil influx at 6 h that was markedly increased in the capsaicin-pretreated animals compared to control group. The counts of eosinophils and mononuclear cells in the pleural exudates did not differ significantly between capsaicin and control groups. The increased levels of immunoglobulin (Ig)E, IgG1 and IgG2a anti-ovalbumin antibodies in serum of sensitized rats did not differ between capsaicin and control groups. In conclusion, the exacerbated pulmonary neutrophil recruitment caused by the capsaicin neonatal treatment is unrelated to increase in serum immunoglobulin antibodies, and suggests a protective role for C-fibers in attenuating the allergic neutrophil infiltration.