Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

Abstract

BackgroundRhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity.MethodsThe neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.ResultsVenom extract (50 μg/mL) increased the muscle twitch tension in PND preparations but did not significantly alter the resting membrane potential values. Electrophysiological evaluations showed that the extract (50 μg/mL) significantly augmented the frequency of miniature end-plate potential (from 38 ± 3.5 to 88 ± 15 after 60 minutes; n = 5; p < 0.05) and quantal content (from 128 ± 13 to 272 ± 34 after five minutes; n = 5; p < 0.05). Pretreatment with ouabain (1 μg/mL) for five minutes prevented the increase in quantal content (117 ± 18 and 154 ± 33 after five and 60 minutes, respectively).ConclusionThese results indicate that the methanolic extract of R. schneideri venom acts primarily presynaptically to enhance neurotransmitter release in mouse phrenic-diaphragm preparations.

Methanolic extract of Rhinella schneideri (cururú toad) poison crude cause ultrastructural changes in nerve terminals of mouse phrenic nerve-diaphragm preparations

“…Previous studies showed that the methanolic extract of R. schneideri poison compounds are capable of affecting neurotransmission in avian and mammalian preparations. The recovery of the number of synaptic vesicles after 60 min of incubation with the methanolic extract of R. schneideri granular (parotoid) gland secretion indicates a transient increase in the neurotransmitter release, which is in accordance to the facilitation observed by Rostelato-Ferreira et al (2014) in PND preparations.…”

“…In their study, Rostelato-Ferreira et al (2014) reported that diaphragm preparations that were incubated with R. schneideri methanolic extract (50 µg/mL of granular gland secretion) responded with an increase in quantal content (release of acetylcholine) that was inhibited with a non-toxic concentration of ouabain (1 mg/mL), an inhibitor of the Na + / K + -ATPase, after 5 min prior to incubation, indicating that these effects were prevented through the inhibition of Na + -K + -ATPase activity of muscle motor innervation.…”

“…The amount of 2 g crude poison was extracted in 50 mL of methanol during three days, at room temperature, lyophilized in a SpeedVac centrifuge (Gao et al, 2010) and stored at -20 °C. The lyophilized methanolic extract was dissolved in Tyrode solution prior to use (Rostelato-Ferreira et al, 2014). Animals: Male Swiss mice (25-30 g) were obtained from the Multidisciplinary Center for Biological Investigation (CEMIB/UNICAMP).…”

“…Mouse phrenic nerve-hemidiaphragm preparations (PND) (Bülbring, 1946) were incubated for 60 min with 50 µg/mL of the methanolic extract in Tyrode solution (composition in mM: NaCl 137, KCl 2.7, CaCl 2 1.8, MgCl 2 0.49, NaH 2 PO 4 0.42, NaHCO 3 11.9 and glucose 11.1; pH 7.5 at 37 ºC) and constantly aerated by carbogen (95 % O 2 -5 % CO 2 ), while control samples were exposed to Tyrode alone (n = 3-5 per group) at same conditions. The 50 µg/mL extract concentration was selected because it was shown to induce neuromuscular facilitation in PND preparation (Rostelato-Ferreira et al, 2014).…”

“…Two recent reports, described such effect on avian and mammalian neuromuscular preparations: in the avian chick biventer cervicis preparation, the methanolic extract of R. schneideri poison was shown to act presynaptically by irreversibly blocking acetylcholine release; no co-participation of post-synaptic nicotinic receptors on the neuromuscular blockade was observed (Rostelato- Ferreira, Dal Belo, Cruz-Höfling, Hyslop, & Rodrigues-Simioni, 2011). Interestingly, in in vitro mice hemidiaphragm preparations R. schneideri poison extract elicited increases in the acetylcholine release, facilitating neurotransmission by a mechanism that suggests the involvement of the Na + K + ATPase pump system (Rostelato-Ferreira, Dal Belo, Leite, Hyslop, & Rodrigues-Simioni, 2014).…”