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Background and Purpose 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. Experimental Approach 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. Key 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 and Implications 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.

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Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [ 125 I]-Tx1 binds specifically and with high affinity (K d = 0.36 ± 0.02 nM) to a single, noninteracting (n H = 1.1), low capacity (Bmax 1.1 pmol/mg protein) binding site. In competition experiments using several compounds (including ion channel ligands), only PhTx2 and PhTx3 competed with [ 125 I]-Tx1 for specific binding sites (K 0.5 apparent = 7.50 x 10 -4 g/l and 1.85 x 10 -5 g/l, respectively). PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [ 125 I]-Tx1 binding. Tx1 reduced the [ 3 H]-ACh release evoked by the PhTx2 pool by 33%, but did not affect basal or KCl-induced [ 3 H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (M-Aga IA and IB) and its competition with [ 125 I]-MCono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

A family of 4kDa neurotoxic peptides was purified from venoms of Phoneutria spiders. All have six cysteine residues, and low similarity with other neurotoxins. Three toxins caused moderate inhibition of L-type Ca(2+) channels. The structure of toxin PRTx27C3 was modeled and compared with toxin ADO1. The importance of four residues is suggested.