Structure-function Relationship Studies On The C-terminal Tetra-peptide And Pharmacological Characterization Of Neuropeptide FF (NPFF)

In 1985, neuropeptide FF (NPFF) was initially isolated and sequenced as a morphine-modulating peptide, very recent reports have shown that NPFF-related peptides / NPFF receptors system is a neuropeptide family including two precursors (pro-NPFFA and pro-NPFFB) and two G protein-coupled receptors (NPFF1 and NPFF2 receptors). Moreover, the findings reported to date indicate that NPFF mediates a variety of biological actions, such as pain modulation, opioid tolerance and dependence, thermoregulation, cardiovascular action, gastrointestinal regulation, food intake and endocrine modulation. In the present thesis, NPFF and NPVF, two NPFF-related peptides were encoded by two different precursor genes and showed different affinities towards NPFF1 and NPFF2 receptors, were used as the NPFF agonists to study their effects on the isolated mouse colon and the in vivo biological activities in three classical functional models. Moreover, BIBP3226 was used as an NPFF antagonist to investigate the mechanisms of the pharmacological activities.In isolated mouse distal colon assay, the results show that NPFF and NPVF dose-dependently induced the contractions of mouse distal colon, by inhibition of nitric oxide synthesis and release, which is unrelated to cholinergic pathway and is independent from opioid and nociceptin system. The in vivo pharmacological studies suggest that NPFF and NPVF produce a series of in vivo bioactivities. The thermalregulative, anti-opioid and pressor effects of NPFF are modulated mostly by NPFF receptors. Furthermore, the nonpeptide compound BIBP3226 did not evoke marked physiological responses in vivo, it could be considered as an antagonist in pharmacological researches of the NPFF system.The structure-activity relationship studies of NPFF-related peptides suggest that the C-terminal -RFamide is essential for NPFF receptors activation and/or occupation. To further evaluate the importance of C-terminal modification of neuropeptide FF (NPFF), in the present work, two series of analogues of NPFF were synthesized: (1) the four dansylated NPFF analogues with lipophilic N-terminal modifications, the results obtained from in vitro and in vivo assays reveal that the putative agonists and antagonists induce different pharmacological activities. The findings suggest that the C-terminal Phe of NPFF might be a crucial residue to determine the efficacy. In addition, it is expected that in future the novel analogue dansyl-GSRFamide may be developed as a highly potent agonist to investigate the NPFF system. (2) These analogues of PFRFamide, an NPFF agonist that was derived from the C-terminal tetra-peptide of NPFF, were synthesized and investigated for the pharmacological activities in the experiment of cardiovascular regulation. The present study clearly shows that the replacement of Pro~1 and Phe~2 of PFRFamide do not affect its cardiovascular activities, in contrast, the conformation and electronic feature of aromatic ring of Phe~4 is very critical for the regulation of the cardiovascular actions.