title: PnPP-19, a spider toxin peptide, induces peripheral antinociception through opioid and cannabinoid receptors and inhibition of neutral endopeptidase.
abstract: OBJECTIVE
  The synthetic peptide PnPP-19 has been studied as a new drug candidate to treat erectile dysfunction. However, PnTx2-6, the spider toxin from which the peptide was designed, induces hyperalgesia. Therefore, we intended to investigate the role of PnPP-19 in the nociceptive pathway.
  
  
  METHODS
  Nociceptive thresholds were measured by paw pressure test. PnPP-19 was administered intraplantarly alone or with selective cannabinoid or opioid receptor antagonists. The hydrolysis of PnPP-19 by neutral endopeptidase (NEP) (EC 3.4.24.11), an enzyme that cleaves enkephalin, was monitored by HPLC and the cleavage sites were deduced by LC-MS. Inhibition by PnPP-19 and Leu-enkephalin of NEP enzyme activity was determined spectrofluorimetrically.
  
  
  RESULTS
  PnPP-19 (5, 10 and 20 μg per paw) induced peripheral antinociception in rats. Specific antagonists of μ opioid receptors (clocinnamox), δ opioid receptors (naltrindole) and CB1 receptors (AM251) partly inhibited the antinociceptive effect of PnPP-19. Inhibition of fatty acid amide hydrolase by MAFP or of anandamide uptake by VDM11 enhanced PnPP-19-induced antinociception. NEP cleaved PnPP-19 only after a long incubation, and Ki values of 35.6 ± 1.4 and 14.6 ± 0.44 μmol·L(-1) were determined for PnPP-19 and Leu-enkephalin respectively as inhibitors of NEP activity.
  
  
  CONCLUSIONS
  Antinociception induced by PnPP-19 appears to involve the inhibition of NEP and activation of CB1, μ and δ opioid receptors. Our data provide a greater understanding of the antinociceptive effects of PnPP-19. This peptide could be useful as a new antinociceptive drug candidate.
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: A C N
authors.initials: AC
authors.lastname: Freitas
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.|Departamento de Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: D F
authors.initials: DF
authors.lastname: Pacheco
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: M F M
authors.initials: MF
authors.lastname: Machado
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: A K
authors.initials: AK
authors.lastname: Carmona
authors.affiliation: Departamento de Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: I D G
authors.initials: ID
authors.lastname: Duarte
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: M E
authors.initials: ME
authors.lastname: de Lima
mesh_terms: D000701:Analgesics, Opioid / Q000494:pharmacology*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008297:Male; D015260:Neprilysin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D043884:Receptor, Cannabinoid, CB1 / Q000037:antagonists & inhibitors*; D011957:Receptors, Opioid / Q000378:metabolism*; D013111:Spider Venoms / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D000701:Analgesics, Opioid; D004791:Enzyme Inhibitors; D010455:Peptides; D043884:Receptor, Cannabinoid, CB1; D011957:Receptors, Opioid; D013111:Spider Venoms; D015260:Neprilysin