The Cloning and Characterization of a Three-Finger Toxin Homolog (NXH8) from the Coralsnake Micrurus corallinus That Interacts with Skeletal Muscle Nicotinic Acetylcholine Receptors.

Coralsnakes (Micrurus spp.) are the only elapids found throughout the Americas. They are recognized for their highly neurotoxic venom, which is comprised of a wide variety of toxins, including the stable, low-mass toxins known as three-finger toxins (3FTx). Due to difficulties in venom extraction and availability, research on coralsnake venoms is still very limited when compared to that of other Elapidae snakes like cobras, kraits, and mambas. In this study, two previously described 3FTx from the venom of M. corallinus, NXH1 (3SOC1_MICCO), and NXH8 (3NO48_MICCO) were characterized. Using in silico, in vitro, and ex vivo experiments, the biological activities of these toxins were predicted and evaluated. The results showed that only NXH8 was capable of binding to skeletal muscle cells and modulating the activity of nAChRs in nerve-diaphragm preparations. These effects were antagonized by anti-rNXH8 or antielapidic sera. Sequence analysis revealed that the NXH1 toxin possesses eight cysteine residues and four disulfide bonds, while the NXH8 toxin has a primary structure similar to that of non-conventional 3FTx, with an additional disulfide bond on the first loop. These findings add more information related to the structural diversity present within the 3FTx class, while expanding our understanding of the mechanisms of the toxicity of this coralsnake venom and opening new perspectives for developing more effective therapeutic interventions.

Cardiovascular activity of Micrurus lemniscatus lemniscatus (South American coralsnake) venom.

Envenomation by coralsnakes (Micrurus spp.) is characterized by blockade of peripheral neurotransmission mediated by the presence of α- and ß-neurotoxins. However, little is known about their cardiovascular activity. Micrurus lemniscatus lemniscatus is a coralsnake found in the Amazon basin and occasionally causes envenomation in humans. In this study, we examined the hemodynamic, vascular and atrial responses to M. l. lemniscatus venom. Anesthetized rats were used for hemodynamic and electrocardiogram (ECG) recordings; in vitro experiments were carried out in rat isolated thoracic aorta and atria preparations. In vivo, venom (0.1 and 0.3 mg/kg) caused immediate and persistent hypotension that was maximal within the first minute with both doses being lethal after ~40 and ~20 min, respectively. ECG, heart and respiratory rates were not altered during the transient hypotension phase induced by venom but all altered prior to death. There was no evidence of myonecrosis in cardiac muscle tissue, pulmonary hemorrhage nor thrombosis in anesthetized rats exposed to venom. In vitro, venom (10 µg/ml) did not contract aortic strips nor affected the maximal responses to pre-contraction with phenylephrine (PE, 0.0001-30 µM) in strips with and without endothelium. However, venom (10 µg/ml) relaxed aortic strips with endothelium pre-contracted with PE. In aortic strips pre-contracted with PE, venom prevented acetylcholine (0.0001-30 µM)-induced relaxation in strips with endothelium without affecting relaxation induced by sodium nitroprusside (0.1-100 nM) in strips without endothelium. Venom (30 µg/ml) produced a transient increase of atrial contractile force without affecting atrial rate. Taken together these findings indicate a predominantly vascular action of the venom, most likely involving toxins interacting with muscarinic receptors.

Experimental model for removal of snake venom via hemoperfusion in rats.

OBJECTIVE: To examine the efficiency of hemoperfusion in removing South American rattlesnake (Crotalus durissus terrificus) venom from rats compared with neutralization by antivenom. DESIGN: An exploratory experimental investigation in rats involving the injection of snake venom with or without subsequent hemoperfusion or antivenom administration. SETTING: Basic animal research laboratory in a private university. ANIMALS: Normal, healthy male Wistar rats (0.29-0.40 kg, 3-6 months old) from a commercial breeder. INTERVENTIONS: Four experimental groups of randomly allocated rats (n = 3/group) were studied: Group 1: rats were injected with a single dose of venom (5 mg/kg, IM, in the right thigh) with no other treatment; blood samples were collected minutes before death to determine leukocyte, platelet, and erythrocyte counts; Group 2 (Control): rats underwent hemoperfusion alone for 60 min using a hemoperfusion cartridge designed for protein adsorption (by granulated charcoal) and protein precipitation (by tannic acid); Group 3 (Venom + antivenom): rats were injected with venom (5 mg/kg, IM) and, 10 min later, were treated with antivenom at the venom:antivenom ratio recommended by the manufacturer; Group 4 (Venom + hemoperfusion): Rats were injected with venom (5 mg/kg, IM) and, 10 min later, were hemoperfused for 60 min. In groups 2-4, blood samples were collected for leukocyte, platelet, and erythrocyte counts 24 h after venom. MEASUREMENTS AND MAIN RESULTS: Rats injected with venom alone (Group 1) developed signs of neurotoxicity and ataxia and died in 9.0 ± 0.43 h but showed no changes in leukocyte or erythrocyte counts. In contrast, there were no deaths in groups 2-4. The lack of deaths in Groups 3 and 4 indicated that antivenom and hemoperfusion, respectively, protected against the lethal effects of the venom. CONCLUSIONS: Hemoperfusion with a double-action hemoperfusion cartridge capable of protein adsorption and precipitation protected rats against C. d. terrificus venom.

Pharmacological analysis of hemodynamic responses to Lachesis muta (South American bushmaster) snake venom in anesthetized rats.

In this work, we examined some mechanisms involved in the hypotension caused by Lachesis muta (South American bushmaster) venom in anesthetized rats. Venom (1.5 mg/kg, i.v.) caused immediate hypotension that was maximal after 5 min and gradually returned to baseline over 60 min. Pretreatment of rats with the non-selective nitric oxide synthase (NOS) inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) did not attenuate the early phase of venom-induced hypotension, but abolished the recovery phase and resulted in rapid death; a similar effect was observed with the soluble guanylate cyclase (sGC) inhibitor ODQ. In contrast, the hemodynamic responses to venom were not attenuated by the non-selective NOS inhibitor NG-monomethyl-L-arginine, the inducible NOS inhibitor aminoguanidine, the phosphodiesterase 5 inhibitor sildenafil, the adenylate cyclase (AC) inhibitor SQ-22.536, the non-selective muscarinic receptor antagonist atropine, the bradykinin B2 receptor antagonist HOE-140 and the non-selective cyclooxygenase inhibitor indomethacin. Preincubation of venom with the PLA2 inhibitor pBPB had no effect on the immediate hypotension but tended to improve the recovery phase. Neither AEBSF (a serine proteinase inhibitor) nor EDTA (a metalloproteinase inhibitor) prevented the venom-induced hypotension, but AEBSF and not EDTA protected against the lethality of a high dose (3.0 mg/kg, i.v.). There were no marked changes in the ECG parameters with the various treatments, except with L-NAME and ODQ that increased the RR interval. Pulmonary thrombus formation was markedly enhanced by L-NAME and ODQ, and to a lesser extent by pBPB, especially in small vessels, whereas AEBSF and EDTA inhibited thrombus formation. Venom relaxed phenylephrine-precontracted thoracic aorta and pulmonary artery in vitro, with the latter being more sensitive. The relaxation was endothelium-dependent and was inhibited by ODQ but not by H-89, a protein kinase A (PKA) inhibitor. Together, these findings indicate involvement of the NO/sGC/cGMP, but not the AC/cAMP/PKA signaling pathway, in the hemodynamic responses to L. muta venom in rats. Muscarinic mechanisms, kinins and arachidonic acid metabolites are apparently not involved.

Hemodynamic responses to Lachesis muta (South American bushmaster) snake venom in anesthetized rats.

In this work, we examined the hemodynamic responses to Lachesis muta (South American bushmaster) venom in anesthetized male Wistar rats. Venom (1.5 mg/kg, i.v.) caused immediate hypotension that was followed by a gradual return towards baseline over 60 min; there were no significant changes in heart rate, ECG parameters and respiratory rate. A higher dose (3 mg/kg, i.v.) caused sustained hypotension, variable bradycardia, respiratory depression and fluctuations in ECG; death occurred within 10-60 min. Venom injected intramuscularly (15 mg/kg) produced a smaller decrease in blood pressure that was more persistent than with 1.5 mg/kg (i.v.). Pre-treatment with atenolol (selective ß1-adrenergic receptor antagonist) potentiated the response to venom (1.5 mg/kg, i.v.) and resulted in a hemodynamic profile similar to that seen with 3 mg/kg (i.v.). Macroscopically, systemic hemorrhage was seen only in the ileum, whereas histological analysis revealed extensive pulmonary hemorrhage; the heart, liver and kidney were generally unaffected. Intravascular pulmonary thrombosis occurred with venom given i.v. and i.m., but was less marked with the latter route. In rat isolated perfused hearts, venom caused a persistent decrease in left ventricular developed pressure but no change in heart rate, coronary flow or ECG; there was tissue necrosis and release of CK-MB that were abolished by pre-treating venom with the PLA2 inhibitor p-bromophenacyl bromide. These results show that in rats L. muta venom causes hypotension, bradycardia and respiratory depression, depending on the dose and route of administration. The hemodynamic responses apparently do not involve direct cardiotoxicity and are modulated by the adrenergic system.

Rat atrial responses to Bothrops jararacussu (jararacuçu) snake venom.

Envenoming by the pitviper Bothrops jararacussu produces cardiovascular alterations, including coagulopathy, systemic hemorrhage, hypotension, circulatory shock and renal failure. In this work, we examined the activity of this venom in rat isolated right atria. Incubation with venom (0.025, 0.05, 0.1 and 0.2mg/ml) caused concentration-dependent muscle contracture that was not reversed by washing. Muscle damage was seen histologically and confirmed by quantification of creatine kinase-MB (CK-MB) release. Heating and preincubation of venom with p-bromophenacyl bromide (a phospholipase A2 inhibitor) abolished the venom-induced contracture and muscle damage. In contrast, indomethacin, a non-selective inhibitor of cyclooxygenase, and verapamil, a voltage-gated Ca(2+) channel blocker, did not affect the responses to venom. Preincubation of venom with Bothrops or Bothrops/Crotalus antivenom or the addition of antivenom soon after venom attenuated the venom-induced changes in atrial function and tissue damage. These results indicate that B. jararacussu venom adversely affected rat atrial contractile activity and muscle organization through the action of venom PLA2; these venom-induced alterations were attenuated by antivenom.

Cardiovascular responses to Bothrops alternatus (Urutu) snake venom in anesthetized dogs.

The cardiovascular responses to Bothrops alternatus snake venom in anesthetized dogs were investigated. Venom (0.3 mg/kg, i.v.) markedly decreased arterial blood pressure, coronary perfusion pressure, and cardiac output (CO) after 5 min, with progressive recovery of the first two parameters to pre-venom levels after 3 h; CO showed little recovery. There was an abrupt, sustained decrease in left and right ventricular systolic work and stroke volume but no significant changes in heart rate, electrocardiogram, and pulmonary hemodynamics; systemic vascular resistance increased from 1 h onwards. A venom dose of 1 mg/kg produced more pronounced cardiovascular alterations, with a progressive decrease to death. There were no significant changes in blood gas (pO(2), pCO(2), HCO(3), SBC, and SBE) and metabolic (pH, lactate, glucose, creatine kinase activity, Na(+), and K(+)) parameters, although there was a transitory increase in plasma lactate dehydrogenase 2 min after the lower venom dose. There were no cardiac histological alterations, but microaneurysms and epithelial desquamation were seen in renal tubules. Circulating venom concentrations (determined by ELISA) decreased rapidly after administration, but venom was still detectable after 4 h. These results show that in dogs, B. alternatus venom produces marked hypotension and a direct cardiac action, with few metabolic alterations.