Modulation of neuropeptide FF (NPFF) receptors influences the expression of amphetamine-induced conditioned place preference and amphetamine withdrawal anxiety-like behavior in rats

Many data indicate that endogenous opioid system is involved in amphetamine-induced behavior. Neuropeptide FF (NPFF) possesses opioid-modulating properties. The aim of the present study was to determine whether pharmacological modulation of NPFF receptors modify the expression of amphetamine-induced conditioned place preference (CPP) and amphetamine withdrawal anxiety-like behavior, both processes relevant to drug addiction/abuse. Intracerebroventricular (i.c.v.) injection of NPFF (5, 10, and 20 nmol) inhibited the expression of amphetamine CPP at the doses of 10 and 20 nmol. RF9, the NPFF receptors antagonist, reversed inhibitory effect of NPFF (20 nmol, i.c.v.) at the doses of 10 and 20 nmol and did not show any effect in amphetamine- and saline conditioned rats. Anxiety-like effect of amphetamine withdrawal was measured 24h after the last (14 days) amphetamine (2.5mg/kg, i.p.) treatment in the elevated plus-maze test. Amphetamine withdrawal decreased the percent of time spent by rats in the open arms and the percent of open arms entries. RF9 (5, 10, and 20 nmol, i.c.v.) significantly reversed these anxiety-like effects of amphetamine withdrawal and elevated the percent of time spent by rats in open arms at doses of 5 and 10 nmol, and the percent of open arms entries in all doses used. NPFF (20 nmol) pretreatment inhibited the effect of RF9 (10 nmol). Our results indicated that stimulation or inhibition of NPFF receptors decrease the expression of amphetamine CPP and amphetamine withdrawal anxiety, respectively. These findings may have implications for a better understanding of the processes involved in amphetamine dependence.     

Inhibition of neuropeptide FF (NPFF)-induced hypothermia and anti-morphine analgesia by RF9, a new selective NPFF receptors antagonist

Neuropeptide FF (NPFF) belongs to a neuropeptide family including two precursors (pro-NPFF_A and pro-NPFF_B) and two receptors (NPFF₁ and NPFF₂). Very recently, the novel compound RF9 was reported as the truly selective antagonist on NPFF receptors. The present study examined the effects of RF9 on the hypothermia and anti-morphine action induced by NPFF in mice. (1) RF9 injected into the third ventricle was devoid of any residual agonist activity, but it completely antagonized the hypothermic effects of NPFF (30 or 45 nmol) after cerebral administration in mice; (2) RF9 did not alter the tail-flick latency and morphine analgesia in nociceptive test, however, co-administration of RF9 prevented the anti-morphine action of intracerebroventricularly applied NPFF (10 nmol, i.c.v.) in the mouse tail-flick assay. Collectively, our data indicate that RF9, behaving as a truly pure NPFF receptors antagonist, prevents NPFF-induced drops of the body temperature and morphine analgesia in mice. In addition, it further confirms that the hypothermia and anti-morphine action of NPFF are mediated directly by NPFF receptors.     

Anti-allodynic effects of intrathecally and intracerebroventricularly administered 26RFa, an intrinsic agonist for GRP103, in the rat partial sciatic nerve ligation model

26RFa and QRFP are endogenous ligands of GPR103. 26RFa binding sites are widely distributed in the brain and the spinal cord where they are involved in processing pain. In the present study, the effects of intrathecal and intracerebroventricular applications of 26RFa on the level of mechanical allodynia induced by partial sciatic nerve ligation were examined in rats. The level of mechanical allodynia was measured using von Frey filaments. Intrathecal and intracerebroventricular injection of 26RFa attenuated the level of mechanical allodynia. 26RFa has been reported to activate not only GPR103 but also neuropeptide FF2 receptor and the effect of intrathecally and intracerebroventricularly administered 26RFa was not antagonized by BIBP3226, an antagonist of neuropeptide FF receptor. Immunohistochemical examination revealed that QRFP-like immunoreactivity (QRFP-LI) was expressed mainly in the small to medium sized neurons in the L5 dorsal root ganglion (DRG) and that partial sciatic nerve injury increased the percentage of QRFP-LI positive neurons. 7 days after the nerve injury, QRFP-LI positive neurons in the L5 DRG ipsilateral to the partial sciatic nerve injury were larger than those in the L5 DRG ipsilateral to the sham operation. These data suggest that (1) exogenously applied 26RFa modulates nociceptive transmission at the spinal and the supraspinal brain in the neuropathic pain model, (2) the mechanism 26RFa uses to produce an anti-allodynic effect may be mediated by the activation of GPR103, and (3) partial sciatic nerve ligation affects the expression of QRFP-LI in the dorsal root ganglion.     

Functional properties of Pfr(Tic)amide and BIBP3226 at human neuropeptide FF2 receptors

The functional characteristics of two putative neuropeptide FF (NPFF) antagonists, BIBP3226 and PFR(Tic)amide, on the human neuropeptide FF receptor subtype 2 (hNPFF2) were investigated. Surprisingly, PFR(Tic)amide was shown to exhibit agonist properties in the [³⁵S]guanosine-5′-O-(3-thio)triphosphate ([³⁵S]GTPγS) binding assay. The efficacy of PFR(Tic)amide was significantly greater than that of (1DMe)Y8Fa, a stable analog of NPFF, and PFR(Tic)amide can therefore be classified as a ‘super-agonist’. BIBP3226 did act as a reversible competitive antagonist on the hNPFF2 receptor. However, high concentrations of BIBP3226 also non-specifically increased [³⁵S]GTPγS binding. The usefulness of BIBP3226 as an antagonist tool on the NPFF receptor is thus limited.     

Neuropeptide FF receptor antagonist, RF9, attenuates the fever induced by central injection of LPS in mice

The endogenous opioid system has been found to be involved in the fever caused by lipopolysaccharide (LPS). Neuropeptide FF (NPFF, FLFQPQRF-NH₂) is an endogenous peptide known to modulate opioid activity, mainly in the central nervous system. Therefore, those data suggested a link between LPS-induced fever and NPFF systems. Using a model of acute neuroinflammation, we sought to determine the effects of NPFF systems on the fever induced by i.c.v. injection of LPS. Coinjected with different doses of NPFF (10 and 30 nmol), the fever of LPS (125 ng) was not modified. Interestingly, the selective NPFF receptors antagonist RF9 (30 nmol) injected into the third ventricle failed to induce significant effect, but it decreased the fever of LPS (125 ng) after cerebral administration in mice. These results suggest that NPFF receptors activation is required for LPS to produce fever. This interaction is the first evidence that NPFF systems participate in the control of acute neuroinflammation in conscious animals.     

Multiple Primary Cilia Modulate the Fluid Transcytosis in Choroid Plexus Epithelium

Functional defects in cilia are associated with various human diseases including congenital hydrocephalus. Previous studies suggested that defects in cilia not only disrupt the flow of cerebrospinal fluid (CSF) generated by motile cilia in ependyma lining the brain ventricles, but also cause increased CSF production at the choroid plexus. However, the molecular mechanisms of CSF overproduction by ciliary dysfunction remain elusive. To dissect the molecular mechanisms, choroid plexus epithelial cells (CPECs) were isolated from porcine brain. These cells expressed clusters of primary cilia on the apical surface. Deciliation of CPECs elevated the intracellular cyclic AMP (cAMP) levels and stimulated basolateral‐to‐apical fluid transcytosis, without detrimental effects on other morphological and physiological features. The primary cilia possessed neuropeptide FF (NPFF) receptor 2. In deciliated cells, the responsiveness to NPFF was reduced at nanomolar concentrations. Furthermore, CPECs expressed NPFF precursor along with NPFFR2. An NPFFR antagonist, BIBP3226, increased the fluid transcytosis, suggesting the presence of autocrine NPFF signaling in CPECs for a tonic inhibition of fluid transcytosis. These results suggest that the clusters of primary cilia in CPECs act as a sensitive chemosensor to regulate CSF production.     

Neuropeptide FF activates ERK and NF kappa B signal pathways in differentiated SH-SY5Y cells

Neuropeptide FF (NPFF) has been reported to play important roles in regulating diverse biological processes. However, little attention has been focused on the downstream signal transduction pathway of NPFF. Here, we used the differentiated neuroblastoma cell line, dSH-SY5Y, which endogenously expresses hNPFF2 receptor, to investigate the signal transduction downstream of NPFF. In particular we investigated the regulation of the extracellular signal-regulated protein kinase (ERK) and the nuclear factor kappa B (NF-κB) pathways by NPFF in these cells. NPFF rapidly and transiently stimulated ERK. H89, a selective inhibitor of cyclic AMP-dependent protein kinase A (PKA), inhibited the NPFF-activated ERK pathway, indicating the involvement of PKA in the NPFF-induced ERK activation. Down-regulation of nitric oxide synthases also attenuated NPFF-induced ERK activation, suggesting that a nitric oxide synthase-dependent pathway is involved. Moreover, the core upstream components of the NF-κB pathway were also significantly activated in response to NPFF, suggesting that the NF-κB pathway is involved in the signal transduction pathway of NPFF. Collectively, these data demonstrate that nitric oxide synthases are involved in the signal transduction pathway of NPFF, and provide the first evidence for the interaction between NPFF and the NF-κB pathway. These advances in our interpretation of the NPFF pathway mechanism will aid the comprehensive understanding of its function and provide novel molecular insight for further study of the NPFF system.     

Regulation of μ binding sites after chronic administration of antibodies directed against specific anti-opiate peptides

There is some indication that anti-opiate peptides (AOP) modulate opioid receptor systems by altering μ-receptor density. To further characterize this phenomenon, we investigated the effects of continuous infusion of anti-AOP IgG on μ binding sites in the brains of rats. Specifically, male Sprague–Dawley rats received intracerebroventricular (ICV) infusions for 13 days of either control (rabbit) IgG or test IgGs: anti-dynorphin A IgG, anti-dynorphin A_1–8 IgG, anti-α-MSH IgG, or the monoclonal anti-NPFF IgG. Administration of anti-NPFF IgG or the anti-dynorphin_1–8 IgG significantly increased μ labeling by 40–70% in several brain regions at the caudate level. Contrary to these findings, anti-α-MSH IgG decreased (19–32%) [¹²⁵I]-DAMGO labeling in several thalamic nuclei. The results suggest that the density of μ-opioid receptors is regulated in part by anti-opiate peptides in the extracellular fluid of the brain.     

Neuropeptide FF-sensitive confinement of mu opioid receptor does not involve lipid rafts in SH-SY5Y cells

*

Mu opioid (MOP) receptor activation can be functionally modulated by stimulation of Neuropeptide FF 2 (NPFF₂) G protein-coupled receptors. Fluorescence recovery after photobleaching experiments have shown that activation of the NPFF₂ receptor dramatically reduces the fraction of MOP receptors confined in microdomains of the plasma membrane of SH-SY5Y neuroblastoma cells. The aim of the present work was to assess if the direct observation of receptor compartmentation by fluorescence techniques in living cells could be related to indirect estimation of receptor partitioning in lipid rafts after biochemical fractionation of the cell. Our results show that MOP receptor distribution in lipid rafts is highly dependent upon the method of purification, questioning the interpretation of previous data regarding MOP receptor compartmentation. Moreover, the NPFF analogue 1DMe does not modify the distribution profile of MOP receptors, clearly demonstrating that membrane fractionation data do not correlate with direct measurement of receptor compartmentation in living cells.     

Neuropeptide FF (NPFF) reduces the expression of cocaine-induced conditioned place preference and cocaine-induced sensitization in animals

The endogenous brain opioid system is believed to play an important role in mediating reward mechanisms. Opioid innervation is high in many limbic regions and reinforcing actions of many drugs of abuse, including cocaine, are thought to be mediated via endogenous opioid system. The aim of the present study was to indicate whether the anti-opioid peptide, neuropeptide FF (NPFF; FLFQPQRF-NH₂) was able to modify the rewarding effect of cocaine (5 mg/kg) measured in the expression of conditioned place preference (CPP) test in rats and the expression of sensitization to hyperlocomotor effect of cocaine (10 mg/kg) in mice. Our results indicate that NPFF (5, 10, and 20 nmol) given intracerebroventricularly (i.c.v.) inhibited the expression of cocaine-induced CPP at the dose of 10 nmol (P < 0.01) and 20 nmol (P < 0.001). Moreover, NPFF inhibited the expression of cocaine-induced sensitization to its hyperlocomotor effect at the dose of 20 nmol (P < 0.05) and acute hyperlocomotor effect of cocaine at doses of 5 nmol (P < 0.01), 10 nmol (P < 0.01), and 20 nmol (P < 0.05). Our study suggests that NPFF may participate in a rewarding effect of cocaine measured in the CPP paradigm. On the other hand, our experiments indicate that NPFF is involved in the mechanism of expression of sensitization to cocaine hyperlocomotion but this effect seems to be non-specific because NPFF also inhibited the acute hyperlocomotor effect of cocaine.