Studies Of The Pharmacological Activities Of Neuropeptide FF Receptors And Their Mechanisms

Neuropeptide FF (NPFF) is an endogenous Arg-Phe-NH2 terminated octapeptide, which has similar C-terminal sequence with the molluscan cardioexcitory peptide FMRF-NH₂ belongs to the FMRF-NH₂ related neuropeptide family. NPFF was first isolated and sequenced from bovine brain. Shortly thereafter, two G protein-coupled receptors, NPFF1 and NPFF2 were cloned. NPFF1 receptors are primarily shown to be localized in the supraspinal regions, while NPFF2 receptors are restricted to the brain and spinal cord. Only a few NPFF receptors were detected on the peripheral level. NPFF related peptides were deprived from two precursors, pro-NPFFA and pro-NPFFB. Pro-NPFFA contains NPFF, and NPVF is generated from pro-NPFFB. NPFF has been found to be selective for NPFF2 receptors, but NPVF has highly affinities toward NPFF1 receptors. NPFF modulates opioid induced analgesia and is so called morphine-modulating peptide. NPFF is involved in modulating a variety of biological actions, such as nociceptive, thermal, cardiovascular, gastrointestinal, food intake and hormone modulation. NPFF and NPVF were synthesized by solid phase peptide synthesis method. The present thesis is designed to study the effects of NPFF and NPVF on the gastrointestinal system and other in vivo biological activities.NPFF receptors exert effects on the in vitro gastrointestinal system. The bioactivities and mechanisms of NPFF and NPVF were investigated by isolated mouse distal colon assay. The experiment results show that NPFF and NPVF (1-15 μM) dose-dependently induced the contractions of mouse distal colon, which were abolished by an NPFF putative antagonist, BIBP3226. Cholinergic antagonist atropine or the nonselective opioid antagonist naloxone had no influence on the activities of NPFF and NPVF. NPFF and NPVF did not modify the contractile responses induced by the accumulative doses of acetylcholine or a certain dose of morphine and nociceptin. NPFF and NPVF had no additional effects on the responses evoked by L-NOARG (nitric oxide synthase inhibitor). The contractile responses to NPFF and NPVF were significantly weakened by addition of L-Arg (nitric oxide donor). Theabove experiment results indicate that, NPFF and NPVF could induce isolated mouse distal colon contractions by inhibition of nitric oxide synthesis and release, which is unrelated to cholinergic pathway and is independent from opioid system.NPFF receptors affect the in vivo bioactivities. The in vivo bioactivities of NPFF and NPVF and the inhibitory effects of the putative antagonist BIBP3226 of NPFF receptors were studied by measuring the mouse body temperature, radiant heat tail-flick test, mean arterial pressure and heart rate. In the mouse temperature measurement, 30 nmol of NPFF and NPVF could significantly produced hypothermia, which was prevented by BIBP3226. In the nociceptive test, 10 nmol of NPFF markedly attenuated the analgesia induced by morphine, while BIBP3226 failed to produce significant effects on the antinociceptive responses to morphine. However, the anti-morphine effects of NPFF were prevented by coinjection of BIBP3226. 35 nmol NPVF enhanced the morphine induced analgesia, but BIBP3226 did not modify the responses to NPVF. In the rat hemodynamic experiments, 200 nmol/kg NPFF and NPVF evoked pressor and tachycardiac responses. BIBP3226 significantly inhibited the pressor responses of NPFF and NPVF, but BIBP3226 failed to evidently alter the tachycardia. In the three above in vivo experiments, BIBP3226 did not evoke marked physiological responses. These results suggest that NPFF and NPVF produce a series of bioactivities. The thermalregulative, anti-opioid and pressor effects of NPFF are modulated mostly by NPFF receptors.