Léa Rodrigues Simioni

Neurotoxicity of Micrurus altirostris (Uruguayan coral snake) venom and its neutralization by commercial coral snake antivenom and specific antiserum raised in rabbits

In this work, we studied the neuromuscular blockade caused by Micrurus altirostris venom (0.1–10 μg/mL) in indirect stimulated chick biventer cervicis and mouse phrenic nerve-diaphragm preparations and the ability of commercial antivenom (Instituto Butantan) and antiserum raised in rabbits to neutralize neurotoxicity and lethality in chicks and mice (LD50 0.042 and 0.255 mg/kg), injected i.m. and i.p., respectively, with venom (5 LD50):antivenom or antiserum mixtures (n = 6) of 1:1-1:2.5-1:5-1:10-1:20. The venom caused a complete and irreversible neuromuscular blockade in both preparations, inhibited the acetylcholine and carbachol contractures, without interfering on KCl response. The neuromuscular blockade was not Ca2+ or temperature-dependent and did not affect the response to direct stimulation. Only a venom:antivenom or antiserum ratio of 1:20 neutralized the neuromuscular blockade in vitro and protected chicks and mice against 5 LD50 of venom. Our results indicated that Micrurus altirostris venom interferes with postsynaptic neurotransmission and that commercial antivenom and rabbit antiserum have low efficacy in neutralizing the neurotoxicity and lethality of this venom.

Neuromuscular action of Bothrops lanceolatus (Fer de lance) venom and a caseinolytic fraction

A protein capable of inducing neuromuscular blockade in avian preparations and of depolarizing mouse diaphragm muscle was isolated from Bothrops lanceolatus venom using gel filtration and ion-exchange chromatography. The purified protein was a single chain polypeptide with an estimated molecular mass of 27.5 kDa by SDS-PAGE and had caseinolytic activity (13.3 units/mg), but no phospholipase A(2). B.lanceolatus venom (50 micro g/ml) and the caseinolytic protein (20 micro g/ml) produced contracture and progressive irreversible blockade (50% in 25+/-5 min (SEM) and 45+/-15 min, respectively), in indirectly stimulated chick biventer cervicis preparations. The contractile responses to acetylcholine (ACh; 37 and 74 micro M, n=6) were inhibited by venom and the caseinolytic protein, whereas those to potassium (13.4mM, n=6) were not. Membrane resting potential measurements in mouse hemidiaphragm preparations showed that B.lanceolatus venom and the purified protein caused depolarization which was prevented by D-tubocurarine (14.6mM). The venom produced a slight increase in the amplitude and frequency of miniature end-plate potentials, but this effect was not seen with the purified fraction. These results suggest that the purified protein acts exclusively post-synaptically.

Phenobarbital influence on neuromuscular block produced by rocuronium in rats

PURPOSE To evaluate in vitro and in vivo neuromuscular blockade produced by rocuronium in rats treated with Phenobarbital and to determine cytochrome P450 and cytochrome b5 concentrations in hepatic microsomes. METHODS Thirty rats were included in the study and distributed into 6 groups of 5 animals each. Rats were treated for seven days with phenobarbital (20 mg/kg) and the following parameters were evaluated: 1) the amplitude of muscle response in the preparation of rats exposed to phenobarbital; 2) rocuronium effect on rat preparation exposed or not to phenobarbital; 3) concentrations of cytochrome P450 and cytochrome b5 in hepatic microsomes isolated from rats exposed or not to phenobarbital. The concentration and dose of rocuronium used in vitro and in vivo experiments were 4 microg/mL and 0,6 mg/kg, respectively. RESULTS Phenobarbital in vitro and in vivo did not alter the amplitude of muscle response. The neuromuscular blockade in vitro produced by rocuronium was significantly different (p=0.019) between exposed (20%) and not exposed (60%) rats; the blockade in vivo was significantly greater (p=0.0081) in treated rats (93.4%). The enzymatic concentrations were significantly greater in rats exposed to phenobarbital. CONCLUSIONS Phenobarbital alone did not compromise neuromuscular transmission. It produced enzymatic induction, and neuromuscular blockade in vivo produced by rocuronium was potentiated by phenobarbital.

Exploring and understanding the functional role, and biochemical and structural characteristics of an acidic phospholipase A2, AplTx-I, purified from Agkistrodon piscivorus leucostoma snake venom

ABSTRACT Phospholipases A2 (PLA2s) constitute a class of extensively studied toxins, isolated from snake venoms. Basic PLA2 isoforms mediate various toxicological effects, while the acidic isoforms generally have higher enzymatic activities, but do not promote evident toxic effects. The functions of these acidic isoforms in snake venoms are still not completely understood and more studies are needed to characterize the biological functions and diversification of acidic toxins in order to justify their abundant presence in these secretions. Recently, Lomonte and collaborators demonstrated, in a proteomic and toxicological study, high concentrations of PLA2s in the venom of Agkistrodon piscivorus leucostoma. We have, herein, purified and characterized an acidic PLA2 from this snake venom, denominated AplTx‐I, in order to better understand its biochemical and structural characteristics, as well as its biological effects. AplTx‐I was purified using two chromatographic steps, in association with enzymatic and biological assays. The acidic toxin was found to be one of the most abundant proteins in the venom of A. p. leucostoma; the protein was monomeric with a molecular mass of 13,885.8 Da, as identified by mass spectrometry ESI‐TOF and electrophoresis. The toxin has similar primary and tridimensional structures to those of other acidic PLA2s, a theoretical and experimental isoelectric point of ≈5.12, and a calcium‐dependent enzyme activity of 25.8985 nM/min/mg, with maximum values at 37 °C and pH 8.0. Despite its high enzymatic activity on synthetic substrate, AplTx‐I did not induce high or significant myotoxic, coagulant, anticoagulant, edema, neuromuscular toxicity in mouse phrenic nerve‐diaphragm preparations or antibacterial activities. Interestingly, AplTx‐I triggered a high and selective neuromuscular toxicity in chick biventer cervicis preparations. These findings are relevant to provide a deeper understanding of the pharmacology, role and diversification of acidic phospholipase A2 isoforms in snake venoms. HIGHLIGHTSAplTx‐I is an acidic and monomeric phospholipase A2, isolated from Agkistrodon piscivorus leucostoma venom.AplTx‐I has high catalytic activity on synthetic and non‐micellar substrate.The amino acid sequence of AplTx‐I was identified by mass spectrometry.In vitro and in vivo, AplTx‐I presented low‐level, or lack of, toxicity to mammals and bacteria.AplTx‐I promoted an irreversible and selective neuromuscular blockade in biventer cervicis preparations.

In vitro and in vivo neuromuscular effects of atracurium and rocuronium in rats treated with carbamazepine for seven days

JUSTIFICATIVA Y OBJETIVOS: Se trata de un estudio experimental que investigo in vitro e in vivo el bloqueo neuromuscular producido por el rocuronio y atracurio en ratones tratados con carbamazepina y determino las concentraciones de citocromo P450 y b5 reductasis en microsomas hepaticos. METODO: Ratones fueron tratados por siete dias con carbamazepina (CBZ) - 40 mg.kg-1 a traves de una sonda y sacrificados al octavo dia bajo anestesia con uretana. Las preparaciones in vitro e in vivo fueron montadas de acuerdo con las tecnicas de Bulbring y de Leeuwin y Wolters, respectivamente. Las concentraciones y dosis utilizadas de los bloqueadores en las preparaciones in vitro e in vivo fueron, respectivamente, 20 µg.mL-1 y 0,5 mg.kg-1 para atracurio (ATC); 4 µg.mL-1 y 0,6 mg.kg-1 para rocuronio (ROC). Cada protocolo tuvo un n = 5 y las respuestas fueron observadas por 60 minutos. Los efectos del ATC y ROC fueron evaluados en las preparaciones de ratones tratados (Cbzt) y comparados a los observados en los de ratones no tratados (CBZst). Las concentraciones de citocromo P450 y b5 reductasis fueron determinadas en microsomas aislados de higados de ratones tratados (CBZt) y comparadas con las obtenidas en ratones no tratados (CBZst) RESULTADOS: La carbamazepina no altero la amplitud de las respuestas musculares; in vitro y in vivo, no hubo diferencia entre el bloqueo neuromuscular producido por el atracurio en las preparaciones CBZt versus CBZst; el bloqueo neuromuscular producido por el Rocuronio en las preparaciones CBZt fue potenciado in vitro. La carbamazepina no altero las concentraciones de citocromo P450 y b5. CONCLUSIONES: El tratamiento por siete dias con carbamazepina, no influencio en el bloqueo producido por el atracurio, y altero in vitro los efectos del rocuronio. El tiempo de tratamiento no fue suficiente para causar la induccion enzimatica y disminuir la sensibilidad al rocuronio.BACKGROUND AND OBJECTIVES This experimental study investigated the in vitro and in vivo neuromuscular blockade of rocuronium and atracurium in rats treated with carbamazepine and determined the concentration of cytochrome P450 and b5 reductase in hepatic microsomes. METHODS Rats were treated with carbamazepine (CBZ)--40 mg x kg(-1) by gavage and sacrificed on the eighth day under anesthesia with urethane. In vitro and in vivo preparations followed the techniques of Bulbring and Leeuwin and Wolters, respectively. Concentrations and doses of the neuromuscular blockers used in in vitro and in vivo preparations were, respectively, 20 microg x mL(-1) and 0.5 mg x kg(-1) for atracurium (ATC); and 4 microg x kg(-1) and 0.6 mg x kg(-1) for rocuronium (ROC). Each protocol had an n = 5 and the response was observed for 60 minutes. The effects of ATC and ROC were evaluated in the preparations of rats treated with carbamazepine (CBZ(t)) and compared to those of non-treated rats (CBZ(st)). The concentration of cytochrome P450 and b5 reductase were determined in hepatic chromosomes of rats treated with carbamazepine (CBZ(t)) and non-treated rats (CBZ(st)). RESULTS Carbamazepine did not change the amplitude of neuromuscular response; differences in the neuromuscular blockade produced by atracurium in CBZ1 preparations were not observed, in vitro or in vivo, when compared with CBZ(st); the neuromuscular blockade produced by rocuronium in CBZ(t) preparations was potentiated in vitro. Carbamazepine did not change the concentrations of cytochrome P450 and b5. CONCLUSIONS Seven-day treatment with carbamazepine did not change the neuromuscular blockade produce by atracurium, but altered the in vitro effects of rocuronium. The duration of the treatment was not enough to cause enzymatic induction and decrease the sensitivity to rocuronium.

Efeitos neuromusculares in vitro e in vivo do atracúrio e do rocurônio em ratos submetidos a tratamento de sete dias com carbamazepina

JUSTIFICATIVA Y OBJETIVOS: Se trata de un estudio experimental que investigo in vitro e in vivo el bloqueo neuromuscular producido por el rocuronio y atracurio en ratones tratados con carbamazepina y determino las concentraciones de citocromo P450 y b5 reductasis en microsomas hepaticos. METODO: Ratones fueron tratados por siete dias con carbamazepina (CBZ) - 40 mg.kg-1 a traves de una sonda y sacrificados al octavo dia bajo anestesia con uretana. Las preparaciones in vitro e in vivo fueron montadas de acuerdo con las tecnicas de Bulbring y de Leeuwin y Wolters, respectivamente. Las concentraciones y dosis utilizadas de los bloqueadores en las preparaciones in vitro e in vivo fueron, respectivamente, 20 µg.mL-1 y 0,5 mg.kg-1 para atracurio (ATC); 4 µg.mL-1 y 0,6 mg.kg-1 para rocuronio (ROC). Cada protocolo tuvo un n = 5 y las respuestas fueron observadas por 60 minutos. Los efectos del ATC y ROC fueron evaluados en las preparaciones de ratones tratados (Cbzt) y comparados a los observados en los de ratones no tratados (CBZst). Las concentraciones de citocromo P450 y b5 reductasis fueron determinadas en microsomas aislados de higados de ratones tratados (CBZt) y comparadas con las obtenidas en ratones no tratados (CBZst) RESULTADOS: La carbamazepina no altero la amplitud de las respuestas musculares; in vitro y in vivo, no hubo diferencia entre el bloqueo neuromuscular producido por el atracurio en las preparaciones CBZt versus CBZst; el bloqueo neuromuscular producido por el Rocuronio en las preparaciones CBZt fue potenciado in vitro. La carbamazepina no altero las concentraciones de citocromo P450 y b5. CONCLUSIONES: El tratamiento por siete dias con carbamazepina, no influencio en el bloqueo producido por el atracurio, y altero in vitro los efectos del rocuronio. El tiempo de tratamiento no fue suficiente para causar la induccion enzimatica y disminuir la sensibilidad al rocuronio.BACKGROUND AND OBJECTIVES This experimental study investigated the in vitro and in vivo neuromuscular blockade of rocuronium and atracurium in rats treated with carbamazepine and determined the concentration of cytochrome P450 and b5 reductase in hepatic microsomes. METHODS Rats were treated with carbamazepine (CBZ)--40 mg x kg(-1) by gavage and sacrificed on the eighth day under anesthesia with urethane. In vitro and in vivo preparations followed the techniques of Bulbring and Leeuwin and Wolters, respectively. Concentrations and doses of the neuromuscular blockers used in in vitro and in vivo preparations were, respectively, 20 microg x mL(-1) and 0.5 mg x kg(-1) for atracurium (ATC); and 4 microg x kg(-1) and 0.6 mg x kg(-1) for rocuronium (ROC). Each protocol had an n = 5 and the response was observed for 60 minutes. The effects of ATC and ROC were evaluated in the preparations of rats treated with carbamazepine (CBZ(t)) and compared to those of non-treated rats (CBZ(st)). The concentration of cytochrome P450 and b5 reductase were determined in hepatic chromosomes of rats treated with carbamazepine (CBZ(t)) and non-treated rats (CBZ(st)). RESULTS Carbamazepine did not change the amplitude of neuromuscular response; differences in the neuromuscular blockade produced by atracurium in CBZ1 preparations were not observed, in vitro or in vivo, when compared with CBZ(st); the neuromuscular blockade produced by rocuronium in CBZ(t) preparations was potentiated in vitro. Carbamazepine did not change the concentrations of cytochrome P450 and b5. CONCLUSIONS Seven-day treatment with carbamazepine did not change the neuromuscular blockade produce by atracurium, but altered the in vitro effects of rocuronium. The duration of the treatment was not enough to cause enzymatic induction and decrease the sensitivity to rocuronium.

Pre- and post-synaptic actions of McN-A-343 in the neuromuscular junction: myographic and electrophysiologic studies in vitro.

McN-A-343 (McN) concentrations up to 1.0 micrograms/ml increased the amplitude of the indirectly elicited muscular contractions of the isolated rat diaphragm. The effect was accompanied by an increase in both frequency and amplitude of miniature endplate potentials. The latter effects were abolished by tetrodotoxin, thus suggesting that McN increases the permeability of presynaptic membrane to sodium ions. Larger doses of McN block the neurotransmission probably acting at post-synaptic level.

A comparative study of the effects induced by MCN-A-343 and acetylcholine on the isolated toad rectus abdominis☆

McN-A-343 (McN), a non nicotinic ganglionic stimulant, induced slow contractile responses of the toad rectus abdominis. A relaxation was also observed when large doses were added in the presence of a contraction caused by acetylcholine (Ach). The relaxation induced by McN could not be overcome by increasing Ach concentration. Bell-shaped log dose-response curves were obtained for McN. d-Tubocurarine caused an unusual change on these curves, suggesting an indirect action of the agonist. This possibility was corroborated by the fact that hemicholinium, procaine, and cold storage of the muscle caused a marked decrease of the organ sensitivity to McN but not to ACh.

Efectos neuromusculares in vitro e in vivo del atracurio y del rocuronio en ratones sometidos a tratamiento de siete días con carbamazepina

JUSTIFICATIVA Y OBJETIVOS: Se trata de un estudio experimental que investigo in vitro e in vivo el bloqueo neuromuscular producido por el rocuronio y atracurio en ratones tratados con carbamazepina y determino las concentraciones de citocromo P450 y b5 reductasis en microsomas hepaticos. METODO: Ratones fueron tratados por siete dias con carbamazepina (CBZ) - 40 mg.kg-1 a traves de una sonda y sacrificados al octavo dia bajo anestesia con uretana. Las preparaciones in vitro e in vivo fueron montadas de acuerdo con las tecnicas de Bulbring y de Leeuwin y Wolters, respectivamente. Las concentraciones y dosis utilizadas de los bloqueadores en las preparaciones in vitro e in vivo fueron, respectivamente, 20 µg.mL-1 y 0,5 mg.kg-1 para atracurio (ATC); 4 µg.mL-1 y 0,6 mg.kg-1 para rocuronio (ROC). Cada protocolo tuvo un n = 5 y las respuestas fueron observadas por 60 minutos. Los efectos del ATC y ROC fueron evaluados en las preparaciones de ratones tratados (Cbzt) y comparados a los observados en los de ratones no tratados (CBZst). Las concentraciones de citocromo P450 y b5 reductasis fueron determinadas en microsomas aislados de higados de ratones tratados (CBZt) y comparadas con las obtenidas en ratones no tratados (CBZst) RESULTADOS: La carbamazepina no altero la amplitud de las respuestas musculares; in vitro y in vivo, no hubo diferencia entre el bloqueo neuromuscular producido por el atracurio en las preparaciones CBZt versus CBZst; el bloqueo neuromuscular producido por el Rocuronio en las preparaciones CBZt fue potenciado in vitro. La carbamazepina no altero las concentraciones de citocromo P450 y b5. CONCLUSIONES: El tratamiento por siete dias con carbamazepina, no influencio en el bloqueo producido por el atracurio, y altero in vitro los efectos del rocuronio. El tiempo de tratamiento no fue suficiente para causar la induccion enzimatica y disminuir la sensibilidad al rocuronio.BACKGROUND AND OBJECTIVES This experimental study investigated the in vitro and in vivo neuromuscular blockade of rocuronium and atracurium in rats treated with carbamazepine and determined the concentration of cytochrome P450 and b5 reductase in hepatic microsomes. METHODS Rats were treated with carbamazepine (CBZ)--40 mg x kg(-1) by gavage and sacrificed on the eighth day under anesthesia with urethane. In vitro and in vivo preparations followed the techniques of Bulbring and Leeuwin and Wolters, respectively. Concentrations and doses of the neuromuscular blockers used in in vitro and in vivo preparations were, respectively, 20 microg x mL(-1) and 0.5 mg x kg(-1) for atracurium (ATC); and 4 microg x kg(-1) and 0.6 mg x kg(-1) for rocuronium (ROC). Each protocol had an n = 5 and the response was observed for 60 minutes. The effects of ATC and ROC were evaluated in the preparations of rats treated with carbamazepine (CBZ(t)) and compared to those of non-treated rats (CBZ(st)). The concentration of cytochrome P450 and b5 reductase were determined in hepatic chromosomes of rats treated with carbamazepine (CBZ(t)) and non-treated rats (CBZ(st)). RESULTS Carbamazepine did not change the amplitude of neuromuscular response; differences in the neuromuscular blockade produced by atracurium in CBZ1 preparations were not observed, in vitro or in vivo, when compared with CBZ(st); the neuromuscular blockade produced by rocuronium in CBZ(t) preparations was potentiated in vitro. Carbamazepine did not change the concentrations of cytochrome P450 and b5. CONCLUSIONS Seven-day treatment with carbamazepine did not change the neuromuscular blockade produce by atracurium, but altered the in vitro effects of rocuronium. The duration of the treatment was not enough to cause enzymatic induction and decrease the sensitivity to rocuronium.