Presynaptic Activity of an Isolated Fraction from Rhinella schneideri Poison

Abstract

Purpose: Rhinella schneideri is a toad found in many regions of the South America. The poison of the glands has cardiotoxic effect in animals and neuromuscular effects in mice and avian preparation. The purpose of this work was to identify the toxin responsible for the neuromuscular effect in avian and mice neuromuscular preparation.Methods: The methanolic extract from R. schneideri poison was fractioned by reversed phase HPLC. The purity and molecular mass were determined by LC/MS mass spectrometry. Chick biventer cervicis and mouse phrenic-nerve diaphragm were used as neuromuscular preparations to identify the toxin.Results: The purification resulted in 32 fractions, which 4 of them were active in neuromuscular preparation. The toxin of fraction 20 were chosen for better reproducibility of the whole extract activity and its molecular mass was 730.6 Da. The toxin produced facilitation of the muscle contraction followed by a complete neuromuscular blockade in chick biventer cervicis preparation in 90 min without interfering with the exogenous response to ACh and KCl. The quantal content was increased from 128 ± 13 (control) to 216 ± 44 (after 5 min and sustained until 60 min) in the presence of the toxin.Conclusion: In conclusion, our results demonstrated that the neuromuscular action of the poison of Rhinella schneideri is a multitoxin effect. More, the present work first isolated a 730.6 Da toxin that better represent the whole poison neuromuscular effect, to which is attributed a presynaptic action in avian and mouse neuromuscular preparation.

Action of heparin and acetylcholine modulators on the neurotoxicity of the toad Rhinella schneideri (Anura: Bufonidae) in Brazil

“…schneideri poison-induced neuromuscular blockade in avian preparations has been associated to a presynaptic mechanism of action, which is characterized by an absent of muscle responses to exogenous ACh and KCl, low creatine kinase (CK) release and no significant morphological damage, indicating that the functionality of postsynaptic nicotinic receptors and muscle membrane integrity were not affected by the poison (Rostelato-Ferreira et al, 2011). Recently, Rostelato-Ferreira et al, (2018 reported the purification and pharmacological characterization of a non-enzymatic presynaptic active toxin (fraction 20) among a total of thirty-two fractions isolated from R. schneideri poison, with molecular mass determined in 730.6 Da; under low concentration, this toxin induces neuromuscular facilitation followed by complete inhibition of muscle twitches recorded in BC preparations (Rostelato-Ferreira et al, 2018), mimicking the neuromuscular effect produced by R. schneideri poison in the same experimental model (Rostelato-Ferreira et al, 2011). The neuromuscular activity of R. schneideri poison has also been investigated in mouse phrenic nerve diaphragm (PND) preparation, in which producing progressive improvement of the acetylcholine release characterized by an increase in the frequency of miniature end-plate potentials and quantal content of evoked end-plate potentials, with no observation of neuromuscular failure (Rostelato-Ferreira et al, 2014).…”

“…These components, especially those bufadienolides and bufotoxins, have been considered potential therapeutic tools for exhibiting cancer inhibitory activity (Meng et al, 2009), apoptosis suppressive action (Qi et al, 2010) and antimicrobial activity (Tempone et al, 2008). The paratoid gland secretion and their components, e.g., non-enzymatic presynaptic active toxin (730.6 Da) isolated from R. schneideri poison (Rostelato-Ferreira et al, 2018), can induce neurotoxicity characterized by suppression of the motor acetylcholine release (Rostelato-Ferreira, Dal Belo, Cruz-Höfling, Hyslop, & Rodrigues-Simioni, 2011;Rostelato-Ferreira, Dal Belo, Leite, Hyslop, & Rodrigues-Simioni, 2014;Rostelato-Ferreira et al, 2018), including cardiotoxicity consisted in arrhythmia and ventricular failure mostly by antagonizing Na + / K + ATPase of cardiomyocytes (Toledo & Jared, 1995;Sakate & Oliveira, 2000;Gadelha, Lima, Batista, Melo, & Soto-Blanco, 2014;Leal et al, 2020). R. marinus and R. vulgaris are responsible for causing the most cases of envenomation in domestic animals (Sakate & Oliveira, 2000;Sakate & Oliveira, 2001).…”

“…In recent years, several biological properties have been characterized from Rhinella schneideri 'Schneider's toad' poison (Rostelato-Ferreira et al, 2011;Rostelato-Ferreira et al, 2014;Abdelfatah, Lu, Schmeda-Hirschmann, & Efferth, 2019;Leal et al, 2020), a species widely distributed in Brazil (Wake, 2020), including the chemical identification and structural characterization of bufadienolides, e.g., marinobufagenin, bufalin, telocinobufagin, hellebrigenin, 20S21R-epoxymarinobufagin and β-sitosterol, which exhibit cytotoxic activity, modulatory action on the complement cascade, anti-inflammatory activity, inhibitory action of serine protease, presynaptic neuromuscular blocking activity and anticonvulsant action (Cunha-Filho et al, 2010;Anjolette et al, 2015;Sousa-Filho et al, 2016;Shibao, Anjolette, Lopes, & Arantes, 2015;Freitas et al, 2017;Rostelato-Ferreira et al, 2018;Baldo et al, 2019;Zheng et al, 2020).…”

Chemical and Pharmacological Screening of Rhinella icterica (Spix 1824) Toad Parotoid Secretion in Avian Preparations

“…The preparations were stabilized for 20 min prior to testing the fractions that were added in a fixed concentration of 10 µg/mL. The potential influence of RIPS and its fractions on postsynaptic cholinergic nicotinic receptors (indicative of neurotoxicity) and skeletal muscle fibers (indicative of myotoxicity) was assessed by comparing the contractile responses to 110 µM ACh and 24 mM KCl, respectively, before (basal responses) and after incubation with the secretion and fractions [90]. Muscle contractile responses were recorded for 120 min after the addition of RIPS or fractions.…”

Assessment of neuropharmacological potential of low molecular weight components extracted from Rhinella schneideri toad poison

“…Poisoned dogs showed mydriasis, nystagmus and opisthotonus [17,18]. Some studies suggest mechanisms of interaction between bufadienolides and the neuromuscular junction [19,20], causing blockage of synaptic transmission [17,21]. Molecules such as resibufogenin and cinobufagin demonstrated actions on voltagegated potassium channels and resibufogenin also in the voltagegated sodium channels [22,23].…”