Crypteins derived from the neuropeptide FF (NPFF)A precursor display NPFF-like effects in nociceptive tests in mice

J Kotlinska1, E Gibula-Bruzda1, P Suder0,2, M Wasielak0,2, H Raoof0,2, A Bodzon-Kulakowska0,2, J Silberring0,2. 1Medical University, Deparment of Pharmacology and Pharmacodynamics, 20-093 Lublin, Poland, 2AGH University of Science and Technology, Faculty of Materials Science and Ceramics, 30-059 Krakow, Poland

Neuropeptide FF(NPFF) is an endogenous peptide modulating pain perception. NPFF and its analogs elicit nociception and prolong morphine-induced analgesia (pro-opioid effect) after spinal injection but administered supraspinally decrease morphine analgesia and inhibit morphine tolerance (anti-opioid effect) (Roumy and Zajac, 1998). NPFF (FLFQPQRF-NH2), neuropeptide AF (NPAF, AGEGLSSPFWSLAAPQRF-NH2) and neuropeptide SF (NPSF, SLAAPQRF-NH2) are derived from pro-NPFFA precursor. The key-feature of these peptides is common PQRFamidated sequence at their C termini responsible for binding to G protein-coupled receptors, NPFF1 and NPFF2 (Mazarguil et al., 2001). The aim of the study was to exam two other, unknown sequences present in the mouse NPFFA precursor that do not possess the PQRFamidated motif at their C termini. One of these peptides resides between positions 85-99 in the mice pro-NPFFA. This peptide was referred to as neuropeptide SA (NPSA; SAWGSWSKEQLNPQA). Another sequence used in the experiment was N-terminal NPSA fragment, referred to as neuropeptide SS (NPSS; SAWGSWS). These peptides, classified as crypteins, were evaluated in the hot-plate and tail immersion tests in Swiss mice (20-25 g; 8-9 per group) in morphine-induced antinociception as compared to NPFF. Morphine was given intraperitoneally (i.p.) (3.2 mg/kg), immediately before the test. All peptides were injected freehand to the lateral ventricle of the brain (i.c.v.) in 5 μl without anesthesia, according to Haley and McCormick (1957). In the hot-plate test the peptides were given at the dose of 20 nmol and in the tail-immersion test at the dose of 10 nmol, 5 min before morphine. RF9, an antagonist of NPFF receptors, was given i.c.v., 5 min before the peptides to confirm an involvement of NPFF receptors in the observed effects. Both tests were performed for 120 min (at 15 min intervals) after morphine injection. The data (maximum percentage effect) were analyzed by two-way ANOVA followed by Tukey-Kramer post-test. In the hot-plate test, morphine antinociception was significantly attenuated by NPFF and both crypteins during 60 min. of the test and maximal efficacy was generated at 30 min for NPFF(1.51 ± 5.83; P<0.001), NPSA (18.71 ± 3.50; P<0.01) and NPSS (8.19 ± 5.57; P<0.01). RF9 reversed these anti-opioid effects of all peptides (P<0.01). In the tail-immersion test all peptides indicated anti-opioid potency during 60 min. of the test. NPFF achieved maximum anti-morphine efficacy at 15 min. (6.16 ±3.08; P<0.001) but NPSA (7.59 ± 2.48; P<0.001) and NPSS (11.79 ± 3.98; P<0.001) at 30 min. RF9 reversed these effects (P<0.01). Our data show that NPSA and NPSS possess NPFF-like anti-opioid activity in these tests. As crypteins probably do not bind to the NPFF receptors (Zajac JM, personal communication) the mechanism underlying such effects of the newly discovered sequences needs further clarification.

Reference:

Haley TJ, McCormick WG. Br J Pharmacol Chemother 1957;12:12-5.

Mazarguil et al. Peptides 2001; 22: 1471-1478.

Roumy M, Zajac JM. Eur J Pharmacol 1998; 12; 345:1-11.