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

Introduction: Rhinella schneideri is a toad widely distributed in South America and its poison is characterized by inducing cardiotoxicity and neurotoxicity. Objective: In this work, we investigated pharmacological strategies to attenuate the peripheral neurotoxicity induced by R. schneideri poison in avian neuromuscular preparation. Methods: The experiments were carried out using isolated chick biventer cervicis preparation subjected to field stimulation for muscle twitches recordings or exposed to acetylcholine and potassium chloride for contracture responses. Results: Poison (10 μg/ml) produced complete neuromuscular blockade in chick biventer cervicis preparation within approximately 70 min incubation (times for 50 and 90 % blockade: 15 ± 3 min and 40 ± 2 min, respectively; P < 0.05, N= 5); contracture responses to exogenous acetylcholine and KCl were unaffected by poison indicating no specificity with postsynaptic receptors or myotoxicity, respectively. Poison (10 μg/ml)induced neuromuscular blockade was not prevented by heparin (5 and 150 IU/ml) under preor post-treatment conditions. Incubation at low temperature (23-25 °C) abolished the neuromuscular blockade; after raising the temperature to 37 °C, the complete neuromuscular blockade was slightly slower than that seen in preparations directly incubated at 37 °C (times for 50 and 90 % blockade: 23 ± 2 min and 60 ± 2.5 min, respectively; P < 0.05, N= 4). Neostigmine (3.3 μM) did not reverse the neuromuscular blockade in BC preparation whereas 3,4-diaminopyridine (91.6 μM) produced a partial and sustained reversal of the twitch responses (29 ± 7.8 % of maximal reversal reached in approximately 40 min incubation; P < 0.05, N= 4). Conclusions: R. schneideri poison induces potent peripheral neurotoxicity in vitro which can be partially reversible by 3,4-diaminopyridine.


Reagents and poison:
Acetylcholine chloride, neostigmine methylsulfate, 3.4-diaminopyridine and heparin sodium salt were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All salts used to prepare the physiological solutions were of analytical grade. The methanolic extract of R. schneideri poison was provided by Universidade Federal do Pampa (UNIPAMPA, São Gabriel, RS, Brazil) through Dr. Cháriston A. Dal Belo. R. schneideri poison was collected from a single specimen by manual compression from parotid glands and an amount of 2 mg was then included in 50 ml of methanol for three days at room temperature (Gao et al., 2010). The methanolic extract was then lyophilized and maintained under refrigeration until used. Aliquots of the lyophilized extract were dissolved in Krebs solution prior to testing in the neuromuscular preparations.
Animals: This study was approved by an institutional Committee for Ethics in Animal Use (CEUA/UNISO, Protocol no. 096/2016) and the experiments were carried out according to the general ethical guidelines for animal use established by the Brazilian Society of Laboratory Animal Science (SBCAL). Male chicks (4-8 days old, HY-line) were provided by Aviculture Santa Barbara Ltda. (Sorocaba, SP, Brazil) and housed in metal cages with a sawdust substrate at 25 °C on a 12 h light/dark cycle with lights turned on at 6 a.m. The animals had free access to food and water.
Chick biventer cervicis (BC) preparation: Chicks were previously euthanized with isoflurane and the biventer cervicis muscles were removed and mounted under a resting tension of 1 g in a 5 ml organ bath (Panlab, Barcelona, Spain) containing aerated (95 % O 2 and 5 % CO 2 ) Krebs solution (composition, in mM: NaCl 118.7, KCl 4.7, CaCl 2 1.88, KH 2 PO 4 1.17, MgSO 4 1.17, NaHCO 3 25.0 and glucose 11.65, pH 7.5) at 37 °C, as described elsewhere (Ginsborg & Warriner, 1960). The preparations were exposed to a field stimulation (0.1 Hz, 0.2 ms, 4-6 V) with stimuli being delivered from a LE 12406 TC stimulator (Panlab, Barcelona, Spain) and the muscle twitches recorded using an MLT0201 force-displacement transducer coupled to a Quad Bridge Amp and PowerLab 4/35 data acquisition system connected to a LabChart Pro v. 6.0 (ADInstruments Pty Ltd., 2020) software (all from ADInstruments Pty Ltd., Sydney, Australia). Muscle contractures to exogenous acetylcholine (ACh, 110 μM) and potassium chloride (KCl, 20 mM) were evoked in the absence of electric stimulation, before and after treatments with poison (10 mg/ml) and heparin (1-30 ml/ml), to assess the function of postsynaptic nicotinic receptors and muscle membrane integrity, respectively (Harvey et al., 1994); the final muscle contractures to ACh and KCl were expressed as a percentage of the resting state, considered as 100 % of response. After the initial assessments with ACh and KCl, the preparations were extensively washed and electrically stimulated for at least 20 min to allow them a stabilization period before treatments. Muscle twitches were recorded for up to 120 min or until complete blockade; some preparations were maintained in Krebs solution alone for 120 min to obtain muscle response control. Other preparations were incubated at 22-24 °C to assess the influence of temperature on poison-induced (10 mg/ml) neuromuscular blockade. Some pharmacological approaches using 3.4-diaminopyridine (91.6 mM) and neostigmine (3.3 mM) were applied to understand the effects of methanolic extract of R. schneideri poison on the avian peripheral neurotransmission. The heparin neutralizing activity on the R. schneideri poison-induced neuromuscular blockade was assessed under two experimental conditions: 1) pre-treatment-poison (10 mg/ml) was previously pre-incubated with heparin (5-150 IU/ml) for 30 min at 37 °C and then tested in the BC preparations; 2) posttreatment-heparin (1 and 30 ml/ml) was added into the recording bath after 10 min exposure to poison (10 mg/ml). The experiments were carried out using four isolated preparations (N= 4) for every protocol described above.

Statistical analysis:
The results (myographic recordings) were expressed as the mean ± SEM of four isolated preparations from different animals for each experimental protocol. Changes in the twitch-tension responses of BC preparations were considered as a percentage relative to baseline (time zero) values. Statistical comparisons were performed using Student's t-test or ANOVA followed by the Tukey-Kramer test, with P < 0.05 indicating significance. All data analyses were done using Origin 8 SR4 v.8.0951 (Microcal Software Inc., 2020) or Prism 5 (GraphPad Software Inc., 2020) software.
Heparin (5 and 150 IU/ml) has also failed to prevent the R. schneideri poison (10 μg/ml)induced neuromuscular blockade under posttreatment experimental condition with either the lowest or highest concentrations (times for 50 and 90 % blockade: 10 ± 1.5 min and 46 ± 2.5 min (5 IU/ml); 18 ± 2 min and 50 ± 3 min (150 IU/ml), respectively; P < 0.05, N= 4) ( Fig. 2A). The contractures to ACh and KCl were not altered either in those preparations incubated with poison and subjected to posttreatment with 5 and 150 IU of heparin/ml (65 ± 1 (ACh) and 73 ± 1 (KCL) for 5 IU/ml; 112.8 ± 27 (ACh) and 130 ± 30 (KCl) for 150 IU/ml, Unaffected muscle contractures to exogenous ACh and KCl after complete neuromuscular blockade induced by poison alone and treated with heparin. The symbols in section A. and columns in section B. are the mean ± SEM (N= 4). In A. *P < 0.05 compared to control preparations; the superposed (*) represent each point within that time interval. In B. the values were expressed as a percentage of the responses observed in basal contractures recorded before the treatments. N = 4); these values were expressed as a percentage of the basal contractures, considered as 100 % of response (Fig. 2B).
Effect of temperature on the R. schneideri poison-induced neuromuscular blockade in chick BC preparation: The poison (10 μg/ml)-induced neuromuscular blockade was abolished in BC preparations maintained at a low-temperature conditions (23-25 °C) for 60 min incubation; however, raising rapidly the temperature to 37 °C after 60 min incubation (without exchanging the physiological media), the poison produced complete neuromuscular blockade which was slightly slower than that seen with BC preparations directly incubated at 37 °C (times for 50 and 90 % blockade: 15 ± 1.4 vs. 23 ± 2 # min and 40 ± 3.2 vs. 60 ± 2.5 # min for preparations directly incubated at 37 °C and those incubated initially at 23-25 °C and then 37 °C, respectively; P < 0.05 compared to times for 50 and 90 % blockade from those preparations directly incubated at 37 °C, N= 4) (Fig. 3).

DISCUSSION
R. schneideri poison is already known to induce potent blockade of the motor neurotransmission in avian neuromuscular preparations in vitro. The negative modulatory activity induced by R. schneideri poison is preceded by facilitation of the twitch responses, seen with lower concentrations (3 and 10 μg/ml), whilst a highest concentration (30 μg/ml) induces only rapid complete blockade of neuromuscular twitches approximately 20 min, with no evidence of facilitation (Rostelato-Ferreira et al., 2011). Here, we have observed that the methanolic extract of R. schneideri poison (10 μg/ml) produced complete neuromuscular blockade in BC preparations approximately 70 min incubation, with no evidence of facilitation; the reasons for these differences between time for complete neuromuscular blockade and occurrence of facilitation are currently unknown, although they may be attributed to the conditions for extraction and maintenance of the poison and/or its chemical composition due to different amounts of toxic bioactive molecules (Leal et al., 2020).
R. 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).
To examine the role of enzymatic properties in the neuromuscular blockade by R. schneideri poison, experiments in BC preparations were done at 22-24 °C instead of 37 °C. The lower temperature condition is known to attenuate the neuromuscular blockade by Bothrops PLA 2 neurotoxins (Cogo et al., 1998;Ponce-Soto et al., 2009;Floriano et al., 2013;Sucasaca-Monzón et al., 2015). Although there is evidence of the presence of PLA 2 enzymes in the mucous gland secretions of R. schneideri toad (Shibao et al., 2018), the same was not observed in the poison of this species. However, incubation at lower temperatures abolished the neuromuscular blockade in BC preparations while a mild and progressive neuromuscular facilitation was observed in 60 min exposure time. Furthermore, the R. schneideri poison caused complete neuromuscular blockade immediately after raising rapidly the temperature to 37 °C at the end of incubation (60 min) at low temperature. These findings may suggest that there is an enzymatic influence in the R. schneideri poison-induced neuromuscular blockade. In this context, a PLA 2 toxin has been identified in the paratoid gland secretion of a closely related species 'Bufotes viridis' (= Bufo viridis) found in Central Asian (Tashmukhamedov et al., 1994).
Neostigmine (NEO), an acetylcholinesterase inhibitor, failed to counteract the neuromuscular blockade induced by R. schneideri poison in BC preparations, suggesting that postsynaptic nicotinic receptors are not involved in the toxic response by toxins present in this poison. However, 3.4-diaminopyridine (3.4-DAP), an antagonist of voltage-gated potassium channels, produced partial and sustained reversal (approximately 30 %) of the neuromuscular blockade by R. schneideri poison in BC preparations. 3.4-DAP antagonizes motor presynaptic potassium channels prolonging the action potential by delaying the membrane repolarization; this effect prolongs the opening of voltage-dependent calcium channels and consequently increase neurotransmitter release (Floriano et al., 2015;Ojala et al., 2021). The mechanism by which 3.4-DAP reverses the R. schneideri poison-induced neuromuscular blockade is unclear. However, since this poison interferes with presynaptic phases to induce neuromuscular failure (Rostelato-Ferreira et al., 2011), the reversal by 3.4-DAP suggests the presence of some sort of presynaptic neurotoxins in the poison that may affect the calcium homeostasis (Rostelato-Ferreira et al., 2018). In addition, bufadienolides present in R. schneideri poison may be directly related to the neuromuscular failure observed in BC preparations by affecting voltage-gated L-type Ca 2+ channels (Song et al., 2017).
Taken together, the results presented in this work show that R. schneideri poison induces potent neuromuscular blockade in avian isolated preparation without affecting the functionality of postsynaptic nicotinic receptors or muscle membrane integrity, suggesting the involvement of presynaptic neurotoxins in this response. Heparin failed to prevent the toad poison-induced neuromuscular blockade under pre-and post-incubation treatments whereas neostigmine was not able to reverse the blockade; 3.4-diaminopyridine showed to be partially efficient to reverse the poison-induced neuromuscular blockade, being a potential drug candidate to be used as a pharmacological tool in other physiopathological studies.