endomorphin-2 and phenylalanyl-leucyl-phenylalanyl-glutaminyl-prolyl-glutaminyl-arginyl-phenylalaninamide

Mounting evidences indicate the functional interactions between neuropeptide FF (NPFF) and opioids, including the endogenous opioids. In the present work, EN-9, a chimeric peptide containing the functional domains of the endogenous opioid endomorphin-2 (EM-2) and NPFF, was synthesized and pharmacologically characterized. In vitro cAMP assay demonstrated that EN-9 was a multifunctional agonist of κ-opioid, NPFF1 and NPFF2 receptors. In the mouse tail-flick test, intracerebroventricularly (i.c.v.) administration of EN-9 produced significant antinociception with an ED50 value of 13.44 nmol, which lasted longer than that of EM-2. In addition, EN-9 induced potent antinociception after both intravenous (i.v.) and subcutaneous (s.c.) injection. Furthermore, the experiments using the antagonists of opioid and NPFF receptors indicated that the central antinociception of EN-9 was mainly mediated by κ-opioid receptor, independently on NPFF receptors. Notably, the central antinociception of EN-9 was not reduced over a period of 6 days repeated i.c.v. injection. Repeated i.c.v. administration of EN-9 with the NPFF1 and NPFF2 receptors antagonist RF9 resulted in a progressive loss of analgesic potency, consistent with the development of tolerance. Moreover, central administration of EN-9 induced the place conditioning aversion only at a high dose of 60 nmol, but not at low doses. At supraspinal level, only high dose of EN-9 (60 nmol, i.c.v.) inhibited gastrointestinal transit via NPFF receptors. Similarly, systemic administration of EN-9 also inhibited gastrointestinal transit at high doses (10 and 30 mg/kg, i.v.). Taken together, the multifunctional agonist of κ-opioid and NPFF receptors EN-9 produced a potent, non-tolerance forming antinociception with limited side effects.

Topics: Analgesics; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Drug Tolerance; HEK293 Cells; Humans; Injections, Intraventricular; Male; Mice; Oligopeptides; Pain Measurement; Peptide Fragments; Peptides; Receptors, Neuropeptide; Receptors, Opioid, kappa

Neuropeptide FF (NPFF) is known to be an endogenous opioid-modulating peptide. Nevertheless, very few researches focused on the interaction between NPFF and endogenous opioid peptides. In the present study, we have investigated the effects of NPFF system on the supraspinal antinociceptive effects induced by the endogenous µ-opioid receptor agonists, endomorphin-1 (EM-1) and endomorphin-2 (EM-2). In the mouse tail-flick assay, intracerebroventricular injection of EM-1 induced antinociception via µ-opioid receptor while the antinociception of intracerebroventricular injected EM-2 was mediated by both µ- and κ-opioid receptors. In addition, central administration of NPFF significantly reduced EM-1-induced central antinociception, but enhanced EM-2-induced central antinociception. The results using the selective NPFF1 and NPFF2 receptor agonists indicated that the EM-1-modulating action of NPFF was mainly mediated by NPFF2 receptor, while NPFF potentiated EM-2-induecd antinociception via both NPFF1 and NPFF2 receptors. To further investigate the roles of µ- and κ-opioid systems in the opposite effects of NPFF on central antinociception of endomprphins, the µ- and κ-opioid receptors selective agonists DAMGO and U69593, respectively, were used. Our results showed that NPFF could reduce the central antinociception of DAMGO via NPFF2 receptor and enhance the central antinociception of U69593 via both NPFF1 and NPFF2 receptors. Taken together, our data demonstrate that NPFF exerts opposite effects on central antinociception of endomorphins and provide the first evidence that NPFF potentiate antinociception of EM-2, which might result from the interaction between NPFF and κ-opioid systems.

Topics: Adamantane; Animals; Benzeneacetamides; Dipeptides; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Injections, Intraventricular; Male; Mice; Naltrexone; Nociception; Oligopeptides; Pyrrolidines

It has been demonstrated that the endogenous mu opioid (MOP) agonist endomorphin-2 (EM-2) produces conditioned place aversion (CPA) and in contrast, morphine exerts opposite action. Neuropeptide FF (NPFF) was reported to act as a functional antagonist of mu opioid receptor and to exert opioid-modulating activities. The present study examined the influence of NPFF on the rewarding action of EM-2, using the unbiased conditioned place preference (CPP) paradigm. For testing the effect of NPFF on the acquisition of EM-2-induced CPA, NPFF and EM-2 were co-injected on the conditioning days without drug treatment on the followed test day. To explore the effect of NPFF on the expression of EM-2-induced CPA, EM-2 was administered alone on the conditioning days, and NPFF was given 5 min before placement in the CPP apparatus on the test day. The results showed that NPFF (2.5, 5 and 10 nmol, i.c.v.) alone caused little place preference change. However, NPFF dose-dependently reversed the acquisition of CPA induced by 30 nmol EM-2 (i.c.v.). Similarly, the expression of EM-2-induced CPA was also reduced by NPFF. Moreover, the effects of NPFF on the acquisition and the expression of EM-2-induced CPA were completely blocked by the NPFF receptors antagonist RF9 (10 nmol, i.c.v.). However, central injection of NPFF neither changed the locomotor activity nor modified the locomotor action of EM-2. These data provide the first evidence for a functional interaction of the endogenous ligands for NPFF and MOP receptors, and further support an anti-opioid character of NPFF system.

Topics: Animals; Conditioning, Psychological; Mice; Morphine; Oligopeptides; Opioid Peptides; Receptors, Neuropeptide; Receptors, Opioid, mu; Reward

Endomorphin-2 (EM2) is a tetrapeptide with remarkable affinity and selectivity for the mu-opioid receptor. In the present study, we used double-fluorescence and electron microscopic immunocytochemistry to identify subsets of EM2-expressing neurons in dorsal root ganglia and spinal cord dorsal horn of adult rats. Within the lumbar dorsal root ganglia, we found EM2 immunoreactivity mainly in small-to-medium size neurons, most of which co-expressed the neuropeptide substance P (SP). In adult rat L4 dorsal root ganglia, 23.9% of neuronal profiles contained EM2 immunoreactivity and ranged in size from 15 to 36 microM in diameter (mean 24.3 +/- 4.3 microM). Double-labelling experiments with cytochemical markers of dorsal root ganglia neurons showed that approximately 95% of EM2-immunoreactive cell bodies also label with SP antisera, 83% co-express vanilloid receptor subtype 1/capsaicin receptor, and 17% label with isolectin B4, a marker of non-peptide nociceptors. Importantly, EM2 immunostaining persisted in mice with a deletion of the preprotachykinin-A gene that encodes SP. In the lumbar spinal cord dorsal horn, EM2 expression was concentrated in presumptive primary afferent terminals in laminae I and outer II. At the ultrastructural level, electron microscopic double-labelling showed co-localization of EM2 and SP in dense core vesicles of lumbar superficial dorsal horn synaptic terminals. Finally, 2 weeks after sciatic nerve axotomy we observed a greater than 50% reduction in EM2 immunoreactivity in the superficial dorsal horn. We suggest that the very strong anatomical relationship between primary afferent nociceptors that express SP and EM2 underlies an EM2 regulation of SP release via mu-opioid autoreceptors.

Topics: Afferent Pathways; Animals; Axotomy; Biomarkers; Calcitonin Gene-Related Peptide; Cell Size; Functional Laterality; Ganglia, Spinal; Glycoproteins; Immunohistochemistry; Intermediate Filament Proteins; Lectins; Male; Membrane Glycoproteins; Mice; Mice, Knockout; Microscopy, Immunoelectron; Nerve Tissue Proteins; Neurofilament Proteins; Neurons, Afferent; Oligopeptides; Peripherins; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Spinal Cord; Substance P; Versicans

Endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) is a novel endogenous opioid with high affinity and selectivity for the mu-opioid receptor. Immunocytochemical studies have located this peptide in spinal cord, brainstem and selected brain regions. However, there are disagreements regarding its distribution between published reports. Furthermore, the distributions reported for the endomorphins resemble that of neuropeptide FF, suggesting that some of the previous findings might be due to cross-reactivity with the latter substance. In the present study, the distribution of endomorphin-2-immunoreactivity (ir) was examined throughout the entire rat brain using an affinity-purified antiserum that appeared not to cross-react with neuropeptide FF. Endomorphin-2-ir cell somata were most prominent in the hypothalamus and the nucleus of the solitary tract (NTS). Endomorphin-2-ir varicose fibers were observed in such areas as the bed nucleus of the stria terminalis, the septal nuclei, the periaqueductal gray, the locus coeruleus, the lateral parabrachial nucleus, the NTS, and the substantia gelatinosa of the medulla. More modest immunoreactivity was seen in substantia nigra, nucleus raphe magnus, the ventral tegmental area, the pontine nuclei and the amygdala. Fibers were also observed in the ventral cerebellum. Of note was the negligible immunoreactivity in the striatum, a region known to express high levels of mu-opioid receptors. Thus, endomorphin-2-ir was widely, but not uniformly, distributed throughout the central nervous system and was associated largely, but not exclusively, with regions expressing mu-opioid receptors. Based on its distribution, it may have a role in the control of neuroendocrine, cardiovascular and respiratory functions, and mood, feeding, sexual behavior and pain.

Topics: Animals; Antibody Affinity; Brain Chemistry; Cross Reactions; Female; Immunohistochemistry; Male; Microscopy, Confocal; Nerve Fibers; Neurons; Oligopeptides; Rabbits; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu