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Scandinavian Journal of Pain

Official Journal of the Scandinavian Association for the Study of Pain

Editor-in-Chief: Breivik, Harald


CiteScore 2017: 0.84

SCImago Journal Rank (SJR) 2017: 0.401
Source Normalized Impact per Paper (SNIP) 2017: 0.452

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1877-8879
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Volume 17, Issue 1

Issues

PhKv a toxin isolated from the spider venom induces antinociception by inhibition of cholinesterase activating cholinergic system

Flavia Karine Rigo
  • Programa de Pós-Graduação em Ciências da Saúde, Universidade do Extremo Sul Catarinense (UNESC), Criciúma, SC, Brazil
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/ Mateus Fortes Rossato
  • Faculdade de Medicina de Ribeirão Preto USP, Departamento de Farmacologia, Ribeirão Preto, SP, Brazil
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/ Gabriela Trevisan
  • Programa de Pós-Graduação em Ciências da Saúde, Universidade do Extremo Sul Catarinense (UNESC), Criciúma, SC, Brazil
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/ Samira Dal-Toé De Prá
  • Programa de Pós-Graduação em Ciências da Saúde, Universidade do Extremo Sul Catarinense (UNESC), Criciúma, SC, Brazil
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/ Rafael Porto Ineu
  • Programa de Pós-graduação em Ciências Biológicas, Bioquímica Toxicológica, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
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/ Mariane Bernardo Duarte
  • Programa de Pós-Graduação em Ciências da Saúde, Universidade do Extremo Sul Catarinense (UNESC), Criciúma, SC, Brazil
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/ Célio José de Castro Junior
  • Programa de Pós-graduação em Ciências da Saúde, Biomedicina e Medicina, Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte, Grupo Santa Casa de Belo Horizonte, Belo Horizonte, MG, Brazil
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/ Juliano Ferreira
  • Programa de Pós graduação em Farmacologia, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil
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/ Marcus Vinicius Gomez
  • Corresponding author
  • Programa de Pós-graduação em Ciências da Saúde, Biomedicina e Medicina, Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte, Grupo Santa Casa de Belo Horizonte, Belo Horizonte, MG, Brazil
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Published Online: 2017-10-01 | DOI: https://doi.org/10.1016/j.sjpain.2017.09.019

Abstract

Background and aims

Cholinergic agents cause antinociception by mimicking the release of acetylcholine (ACh) from spinal cholinergic nerves. PhKv is a peptide isolated from the venom of the armed spider Phoneutria nigriventer. It has an antiarrythmogenic activity that involves the enhanced release of acetylcholine. The aim of this study was to investigate whether PhKv had an antinociceptive action in mice.

Methods

Male albino Swiss mice (25–35 g) were used in this study. The PhKv toxin was purified from a PhTx3 fraction of the Phoneutria nigriventer spider’s venom. Because of its peptide nature, PhKv is not orally available and it was delivered directly into the central nervous system by an intrathecal (i.t.) route. PhKV on the thermal and mechanical sensitivity was evaluated using plantar test apparatus and the up-and-down method. The analgesic effects of PhKv were studied in neuropathic pain (CCI) and in the peripheral capsicin test. In order to test whether PhKv interfered with the cholinergic system, the mice were pre-treated with atropine (5mg/kg, i.p.) or mecamylamine (0.001 mg/kg, i.p.) and the PhKv toxin (30 pmol/site i.t.) or neostigmine (100 pmol/site) were applied 15 min before the intraplantar capsaicin (1 nmol/paw) administrations. To investigate PhKv action on the AChE activities, was performed in vitro and ex vivo assay for AChE. For the in vitro experiments, mice spinal cord supernatants of tissue homogenates (1 mg/ml) were used as source of AChE activity. The AChE assay was monitored at 37 °C for 10 min in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) at 405 nm.

Results

PhKv (30 and 100pmol/site, i.t.) had no effect on the thermal or mechanical sensitivity thresholds. However, in a chronic constriction injury model of pain, PhKv (10pmol/site, i.t.) caused a robust reduction in mechanical withdrawal with an antinociceptive effect that lasted 4 h. A pretreatment in mice with PhKv (30pmol/site, i.t.) or neostigmine (100pmol/site, i.t.) 15min before an intraplantar injection of capsaicin (1 nmol/paw) caused a maximal antinociceptive effect of 69.5 ± 4.9% and 85 ± 2.5%, respectively. A pretreatment in mice with atropine; 5 mg/kg, i.p. or mecamylamine 0.001 mg/kg, i.p. inhibited a neostigimine and PhKv-induced antinociception, suggesting a cholinergic mechanism. Spinal acetylcholinesterase was inhibited by PhKv with ED50 of 7.6 (4.6–12.6 pmol/site, i.t.). PhKv also inhibited the in vitro AChE activity of spinal cord homogenates with an EC50 of 20.8 (11.6–37.3 nM), shifting the Km value from 0.06 mM to 18.5 mM, characterizing a competitive inhibition of AChE activity by PhKv.

Conclusions

Our findings provide, to our knowledge, the first evidence that PhKv caused inhibition of AChE, it increased the ACh content at the neuronal synapses, leading to an activation of the cholinergic system and an antinociceptive response.

Implications

Studies regarding the nociceptive mechanisms and the identification of potential targets for the treatment of pain have become top priorities. PhKv, by its action of stimulating the cholinergic receptors muscarinic and nicotinic system, reduces pain it may be an alternative for controlling the pain processes.

Keywords: PhKv; Pain; Antinociception; Muscarinic receptors; Nicotinic receptors; Acetylcholinesterase

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About the article

Instituto de Ensino e Pesquisa Santa Casa Belo Horizonte, Rua Domingos Vieira, 590, 30150-240 Belo Horizonte, MG, Brazil


Received: 2017-08-11

Revised: 2017-09-20

Accepted: 2017-09-21

Published Online: 2017-10-01

Published in Print: 2017-10-01


Ethical issues: The experiments were performed in accordance with the current ethical guidelines for the investigation of experimental pain in conscious animals and complying with ARRIVE-guidelines (16)(16).

Conflict of interest: We wish to confirm that all the authors of this manuscript have no conflicts of interest and the manuscript was read and approved by all the authors. We also confirmed that the order of authors listed in the manuscript has been approved by all the authors. The corresponding author is the sole contact for the Editorial process.

Funding This work was supported by REDE 00006–14 FAPEMIG (Minas Gerais State Agency for Research and Development), CNPq (471070/2012–2), CAPES Toxinology (AUX-PE 1444/2011) and FAPEMIG (PPM-00482–15). F.K. Rigo, M.F. Rossato and R.P. Ineu are Postdoctoral Fellows.


Citation Information: Scandinavian Journal of Pain, Volume 17, Issue 1, Pages 203–210, ISSN (Online) 1877-8879, ISSN (Print) 1877-8860, DOI: https://doi.org/10.1016/j.sjpain.2017.09.019.

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