No abstract

No abstract

No abstract

No abstract

1 Neuropeptides FF (NPFF) and AF (NPAF) are involved in pain modulation and opioid tolerance. These peptides were known to act through uncharacterized G protein-coupled receptors (GPCR). We describe here, using an aequorin-based assay as screening tool, that an orphan GPCR, previously designated HLWAR77, is a functional high a nity receptor for NPFF and related peptides. This receptor is further designated as NPFFR. This functional response was prevented by pertussis toxin treatment, demonstrating the involvement of G i family members. 5 SQA-NPFF inhibited forskolin induced cyclic AMP accumulation in recombinant CHO cells in a dose dependent manner. This response was abolished as well by pertussis toxin pre-treatment. 6 RT ± PCR analysis of human tissues mRNA revealed that expression of NPFFR was mainly detected in placenta, thymus and at lower levels in pituitary gland, spleen and testis.

No abstract

The distribution of neuropeptide FF receptors (NPFF(1) and NPFF(2)) was analyzed throughout the central nervous system of rodents (rat, mouse, Octodon degus, and guinea pig), rabbit, and marmoset monkey brains, representing three orders of mammals. Quantitative in vitro receptor autoradiography with [(125)I]EYF ([(125)I]EYWSLAAPQRF-NH(2)) and [(125)I]YVP ([(125)I]YVPNLPQRF-NH(2)) as specific radioligands for NPFF(2) and NPFF(1) receptors, respectively, was used. The NPFF(2) receptor is predominantly expressed in all species, except in the central nervous system of Octodon degus, in which it is undetectable. The density of the NPFF(1) subtype is low in rat and mice, moderate in octodon, rabbit, and monkey, and relatively high in the guinea pig. The present study reveals prominent species differences in the NPFF receptors expression in the brain. The distribution pattern of NPFF(2) receptors in the diencephalon and the superficial layers of the spinal cord is consistent with a hypothesized potential role for NPFF in the modulation of sensory input and opioid analgesia. In contrast, the constant presence of NPFF(1) receptors in the septum, the nucleus of the tractus solitarius, and the hypothalamus suggest its participation in neuroendocrine functions.

To elucidate the mechanism of the cellular antiopioid activity of neuropeptide FF (NPFF), we have transfected the SH-SY5Y neuroblastoma cell line, which expresses -and ␦-opioid receptors, with the human NPFF 2 receptor. The selected clone, SH 2 -D9, expressed high-affinity NPFF 2 receptors in the same range order as -and ␦-opioid receptors (100 -300 fmol/mg of protein channels by opioid agonists. In the presence of carbachol, acting on muscarinic receptors to release Ca 2ϩ from the intracellular stores, deltorphin-I and 1DMe enhanced this release. Preincubation with 1DMe reduced the maximal effect of deltorphin-I by 40%, demonstrating an antiopioid effect in this experimental model for the first time. By using peptides corresponding to the carboxyl terminus of the ␣ i1,2 , ␣ i3 , ␣ o , and ␣ s subunits of G proteins, which specifically uncouple receptors from G proteins, we demonstrated that -opioid and NPFF 2 receptors couple to the four subunits assayed. The Ca 2ϩ release from the intracellular stores by 1DMe resulted from the coupling of NPFF 2 receptors with G␣ o and G␣ i1,2 , whereas the coupling with G␣ s reduced the antiopioid effect of 1DMe in the modulation of N-type channels. This SH 2 -D9 cell line now provides the opportunity to study the interaction between both receptors.Neuropeptide FF (NPFF), FLFQPQRFamide, is representative of a family of mammalian amidated neuropeptides whose precursors pro-NPFF A and pro-NPFF B and G proteincoupled receptors NPFF 1 and NPFF 2 have been recently cloned (Zajac, 2001). Although NPFF does not interact with opioid receptors (Gouardères et al., 1998), a close relationship between neuropeptide FF and opioid systems has been clearly demonstrated in the central nervous system, especially in pain perception (Harrison et al., 1998;Roumy and Zajac, 1998;Panula et al., 1999). For instance, supraspinal injection of NPFF analogs, which has little or no effect in pain tests, decreases morphine-induced analgesia (antiopioid activity, Roumy and Zajac, 1998;Panula et al., 1999), whereas spinal administration induces a naloxone-sensitive analgesia and potentiates morphine-induced analgesia (proopioid activity, Roumy and Zajac, 1998;Panula et al., 1999).In neurons, opioids, including nociceptin, 1) inhibit adenylyl cyclase activity and 2) stimulate inwardly rectifying K ϩ channels and inhibit voltage-dependent Ca 2ϩ currents by activating four types, , ␦, , and Opioid Receptor-Like 1, of opioid receptors (Law et al., 2000). This leads to postsynaptic neuronal inhibition and to presynaptic inhibition of transmitter release, respectively. In contrast to opioids, no data report a direct modulation of K ϩ and Ca 2ϩ conductances by NPFF or analogs but rather describe a blockade of the opioid activity on ionic conductances when cells are pretreated with NPFF. NPFF or analogs, which are inactive by themselves, reverse the opioid-induced inhibition of Ca 2ϩ conductance in NPFF 2 receptor-expressing neurons dissociated from rat dorThis work was supported by the Centre National de l...