Enzymatic degradation of neuropeptide FF and SQA-neuropeptide FF in the mouse brain

Degradation of neuropeptide FF (NPFF) and SQA-neuropeptide FF (SQA-NPFF) by mouse brain sections was investigated by using capillary electrophoresis with UV detection for the separation and the identification of the degradation products. The half disappearance time of SQA-NPFF was 2-fold greater than that of NPFF. NPFF was cleaved preferentially into an inactive metabolite, Gln-Arg-Phe-NH2, in the cerebrum slices. SQA-NPFF was hydrolyzed by an unidentified degrading activity to generate NPFF, and NPFF accounted for a larger part of SQA-NPFF degradation in the hindbrain and cervical spinal cord than in the cerebrum slices. These findings suggest that, depending on the brain regions, NPFF produced from SQA-NPFF could prolong the biologic effects of SQA-NPFF.     

Synthesis of FF8181-A

Both enantiomers of FF8181-A were synthesized through optical resolution from the known Diels-Alder reaction product in 15 steps. The absolute configuration of the natural product was determined to be 1S,5S,5aS,9aS,9bS.     

Synthesis of FF8181-A

Both enantiomers of FF8181-A were synthesized through optical resolution from the known Diels-Alder reaction product in 15 steps. The absolute configuration of the natural product was determined to be 1S,5S,5aS,9aS,9bS.     

Dansyl-PQRamide, a possible neuropeptide FF receptor antagonist, induces conditioned place preference

Neuropeptide FF (NPFF) is an endogenous anti-opioid peptide. NPFF could potentiate the naloxone-precipitated morphine withdrawal syndromes in morphine-dependent rats, indicating the possible involvement of the endogenous NPFF system in opioid analgesia and dependence. The present study was performed to examine the effects of dansyl-PQRamide (dns-PQRa), a putative NPFF antagonist, on conditioned place preference (CPP), in addition, its interaction with the opioid system. Two CPP experiments were conducted. First, rats were treated with dns-PQRa (4-13 mg/kg, i.p.) and paired with the non-preferred compartment while the vehicle was paired with the preferred compartment. Second, similar to experiment 1 except naloxone (1 mg/kg, i.p.) was given 10 min prior to each dns-PQRa administration. The post-drug place preference was examined after 4 alternative pairings. Another group of animals after repetitive dns-PQRa treatments were analyzed for levels of neurotransmitters in discrete brain areas. Dns-PQRa (4-13 mg/kg, i.p.) induced a significant dose-dependent CPP. The dns-PQRa-induced CPP was completely blocked by pretreatment with 1 mg/kg i.p. naloxone, while naloxone alone did not induce any place aversion. The chronic dns-PQRa-treated (13 mg/kg, i.p., b.i.d.) rats caused a significant increase in 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in the olfactory tubercle compared to the vehicle-treated controls. There was also an increase in the turnover of serotonin in the olfactory tubercle, nucleus accumbens and medial prefrontal cortex. These results suggest that blockade of the NPFF system produces rewarding, possibly via an inhibition of the anti-opioid action of NPFF. These results also reveal a close relationship between NPFF, drug rewarding and the dopaminergic and serotoninergic neurons in the mesolimbic system.     

Neuropeptide FF receptors antagonist, RF9, attenuates opioid-evoked hypothermia in mice

The present study used the endpoint of hypothermia to investigate opioid and neuropeptide FF (NPFF) interactions in conscious animals. Both opioid and NPFF systems played important roles in thermoregulation, which suggested a link between opioid receptors and NPFF receptors in the production of hypothermia. Therefore, we designed a study to investigate the relationship between opioid and NPFF in control of thermoregulation in mice. The selective NPFF receptors antagonist RF9 (30nmol) injected into the third ventricle failed to induce significant effect, but it completely antagonized the hypothermia of NPFF (45 nmol) after cerebral administration in mice. In addition, RF9 (30 nmol) co-injected i.c.v. in the third ventricle reduced the hypothermia induced by morphine (5nmol,) or nociceptin/orphanin FQ (N/OFQ) (2 nmol). Neither the classical opioid receptors antagonist naloxone (10 nmol) nor NOP receptor antagonist [Nphe(1)]NC(1-13)NH(2) (7.5 nmol) reduced the hypothermia induced by the central injection of NPFF at dose of 45 nmol. Co-injected with a low dose of NPFF (5 nmol), the hypothermia of morphine (5 nmol) or N/OFQ (2 nmol) was not modified. These results suggest that NPFF receptors activation is required for opioid to produce hypothermia. In contrast, NPFF-induced hypothermia is mainly mediated by its own receptors, independent of opioid receptors in the mouse brain. This interaction, quantitated in the present study, is the first evidence that NPFF receptors mediate opioid-induced hypothermia in conscious animals.     

Structure-activity relationships of neuropeptide FF: role of C-terminal regions

A structure-activity study was carried out to determine the importance of the C-terminal amino acids of the octapeptide Neuropeptide FF (NPFF) in binding and agonistic activity. Affinities of NPFF analogues were tested toward NPFF receptors of the rat spinal cord and the human NPFF2 receptors transfected in CHO cells. The activities of these analogues were evaluated by their ability to both inhibit adenylate cyclase in NPFF2 receptor transfected CHO cells and to reverse the effect of nociceptin on acutely dissociated rat dorsal raphe neurons. The substitutions of Phenylalanine8 by a tyrosine, phenylglycine or homophenylalanine were deleterious for high affinity. Similarly, the replacement of Arginine7 by a lysine or D.Arginine induces a loss in affinity. The pharmacological characterization showed that the presence of the amidated Phe8 and Arg7 residues are also extremely critical for activation of anti-opioid effects on dorsal raphe neurons. The sequence of the C-terminal dipeptide seems also to be responsible for the high affinity and the activity on human NPFF2 receptors. The results support the view that a code messaging the molecular interaction toward NPFF-receptors is expressed in the C-terminal region of these peptides but the N-terminal segment is important to gain very high affinity.