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

 

Mateus Amaral Baldo^1 4, Alexandra Olimpio Siqueira Cunha², Lívea Dornela Godoy^2 3, José Luiz Liberato^2 3, Juliana Sakamoto Yoneda⁵, Elisa Correa Fornari-Baldo^1 4, Pietro Ciancaglini⁵, Wagner Ferreira dos Santos^2 3, Eliane Candiani Arantes¹

 

1 Department of Physics and Chemistry, Ribeirão Preto College of Pharmaceutical Science, University of São Paulo, Ribeirão Preto, SP, Brazil

2 Neurobiology and Venoms Laboratory, Department of Biology, Faculty of Philosophy, Sciences and Letters at Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

3 Institute of Neurosciences & Behavior - INeC, Campus USP, Ribeirão Preto, SP, Brazil

4 Health and Science Institute, Paulista University, São Paulo, SP, Brazil

5 Department of Chemistry, Faculty of Philosophy, Sciences and Letters at Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

 

ABSTRACT

Background:

Studies on toad poison are relevant since they are considered a good source of toxins that act on different biological systems. Among the molecules found in the toad poison, it can be highlighted the cardiotonic heterosides, which have a known mechanism that inhibit Na⁺/K⁺-ATPase enzyme. However, these poisons have many other molecules that may have important biological actions. Therefore, this work evaluated the action of the low molecular weight components from Rhinella schneideri toad poison on Na⁺/K⁺-ATPase and their anticonvulsive and / or neurotoxic effects, in order to detect molecules with actions of biotechnological interest.

 

Methods:

Rhinella schneideri toad (male and female) poison was collected by pressuring their parotoid glands and immediately dried and stored at -20 °C. The poison was dialysed and the water containing the low molecular mass molecules (< 8 kDa) that permeate the dialysis membrane was collected, frozen and lyophilized, resulting in the sample used in the assays, named low molecular weight fraction (LMWF). Na⁺/K⁺ ATPase was isolated from rabbit kidneys and enzyme activity assays performed by the quantification of phosphate released due to enzyme activity in the presence of LMWF (1.0; 10; 50 and 100 µg/mL) from Rhinella schneideri poison. Evaluation of the L-Glutamate (L-Glu) excitatory amino acid uptake in brain-cortical synaptosomes of Wistar rats was performed using [3H]L-glutamate and different concentration of LMWF (10^-5 to 10 µg/µL). Anticonvulsant assays were performed using pentylenetetrazole (PTZ) and N-methyl-D-aspartate (NMDA) to induce seizures in Wistar rats (n= 6), which were cannulated in the lateral ventricle and treated with different concentration of LMWF (0.25; 0.5; 1.0; 2.0; 3.0 and 4.0 µg/µL) 15 min prior to the injection of the seizure agent.

 

Results:

LMWF induced a concentration-dependent inhibition of Na⁺/K⁺-ATPase (IC_50% = 107.5 μg/mL). The poison induces an increased uptake of the amino acid L-glutamate in brain-cortical synaptosomes of Wistar rats. This increase in the L-glutamate uptake was observed mainly at the lowest concentrations tested (10^-5 to 10^-2 µg/µL). In addition, this fraction showed a very relevant central neuroprotection on seizures induced by PTZ and NMDA.

 

Conclusions:

LMWF from Rhinella schneideri poison has low molecular weight compounds, which were able to inhibit Na⁺/K⁺-ATPase activity, increase the L-glutamate uptake and reduced seizures induced by PTZ and NMDA. These results showed that LMWF is a rich source of components with biological functions of high medical and scientific interest.

 

Keywords: Rhinella schneideri; toad poison; bufadienolides; seizures; neuroprotection

 

Received: July 31, 2018.

Accepted: October 23, 2018.

 

Correspondence:ecabraga@fcfrp.usp.br

 

Authors' contributions MAB was responsible for carrying out the experiments, extraction and analysis of toad´s poison and writing the manuscript. AOSC conducted seizure experiments, synaptosome assays and data analysis. LDG contributed for writing the manuscript and data analysis. JLL was responsible for experiment design and data analysis. JSY performed all experiments of Na+/K+-ATPase inhibition and data analysis. ECFB assisted in total poison extraction and writing the manuscript. PC supervised the Na+/K+-ATPase inhibition assays, analysis and assisted in the project design. WFS was the major contributor of the project, assisted in the project design and contributed writing the manuscript and data analysis. ECA coordinated the whole team, searched for funding, assisted in the project design and advised MAB. All authors read, corrected and approved the final manuscript.

 

Competing interests The authors declare that there are no competing interests.