fmrfamide and phenylalanyl-leucyl-phenylalanyl-glutaminyl-prolyl-glutaminyl-arginyl-phenylalaninamide

Topics: Animals; FMRFamide; Humans; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Opioid-Related Disorders; Substance Withdrawal Syndrome

The endogenous neuropeptide FF (NPFF) and its two cognate G protein-coupled receptors, Neuropeptide FF Receptors 1 and 2 (NPFFR1 and NPFFR2), represent a relatively new target system for many therapeutic applications including pain regulation, modulation of opioid side effects, drug reward, anxiety, cardiovascular conditions, and other peripheral effects. Since the cloning of NPFFR1 and NPFFR2 in 2000, significant progress has been made to understand their pharmacological roles and interactions with other receptor systems, notably the opioid receptors. A variety of NPFFR ligands with different mechanisms of action (agonists or antagonists) have been discovered although with limited subtype selectivities. Differential pharmacological effects have been observed for many of these NPFFR ligands, depending on assays/models employed and routes of administration. In this Perspective, we highlight the therapeutic potentials, current knowledge gaps, and latest updates of the development of peptidic and small molecule NPFFR ligands as tool compounds and therapeutic candidates.

Topics: Analgesics, Opioid; Animals; Anxiety; Humans; Ligands; Mice; Oligopeptides; Pain; Peptidomimetics; Rats; Receptors, Neuropeptide; Small Molecule Libraries

Gonadotropin-releasing hormone (GnRH) neurons in the terminal nerve (TN) show endogenous pacemaker activity, which is suggested to be dependent on the physiological conditions of the animal. The TN-GnRH neurons have been suggested to function as a neuromodulatory neuron that regulates long-lasting changes in the animal behavior. It has been reported that the TN-GnRH neurons are immunoreactive to FMRFamide. Here, we find that the pacemaker activity of TN-GnRH neuron is inhibited by FMRFamide: bath application of FMRFamide decreased the frequency of pacemaker activity of TN-GnRH neurons in a dose-dependent manner. This decrease was suppressed by a blockage of G protein-coupled receptor pathway by GDP-β-S. In addition, FMRFamide induced an increase in the membrane conductance, and the reversal potential for the FMRFamide-induced current changed according to the changes in [K(+)](out) as predicted from the Nernst equation for K(+). We performed cloning and sequence analysis of the PQRFamide (NPFF/NPAF) gene in the dwarf gourami and found evidence to suggest that FMRFamide-like peptide in TN-GnRH neurons of the dwarf gourami is NPFF. NPFF actually inhibited the pacemaker activity of TN-GnRH neurons, and this inhibition was blocked by RF9, a potent and selective antagonist for mammalian NPFF receptors. These results suggest that the activation of K(+) conductance by FMRFamide-like peptide (≈NPFF) released from TN-GnRH neurons themselves causes the hyperpolarization and then inhibition of pacemaker activity in TN-GnRH neurons. Because TN-GnRH neurons make tight cell clusters in the brain, it is possible that FMRFamide-like peptides released from TN-GnRH neurons negatively regulates the activities of their own (autocrine) and/or neighboring neurons (paracrine).

Topics: Action Potentials; Adamantane; Amino Acid Sequence; Animals; Base Sequence; Biological Clocks; Dipeptides; DNA, Complementary; Dose-Response Relationship, Drug; Female; FMRFamide; Gonadotropin-Releasing Hormone; Guanosine Diphosphate; Ion Transport; Male; Molecular Sequence Data; Neurons; Olfactory Pathways; Oligopeptides; Perciformes; Potassium Channels, Voltage-Gated; Prosencephalon; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Sequence Homology, Amino Acid; Thionucleotides

Neuropeptide FF (NPFF) is an FMRFamide-related peptide widely distributed in the mammalian brain. NPFF immunohistochemistry labeled cell bodies in a few locations and dense fiber networks throughout the brain. Recently, the distribution of NPFF immunoreactive (NPFF-ir) cells and fibers in the brain of anuran and urodele amphibians was studied and, as in mammals, significant species differences were noted. To further assess general and derived features of the NPFF-containing neuron system in amphibians, we have investigated the distribution of NPFF-ir cell bodies and fibers in the brain of the gymnophionan Dermophis mexicanus by means of an antiserum against bovine NPFF. This distribution was compared to that of FMRFamide immunoreactivity. Major traits shared with anurans and urodeles were the abundant fiber labeling in the ventral telencephalon, hypothalamus, isthmus, ventrolateral medulla and dorsal spinal cord. In addition, in the three amphibian orders the majority of the NPFF-ir cells were located in the preoptic-hypothalamic region. However, distinct particular features were present in the gymnophionan such as the lack of NPFF-ir cells in the telencephalon, brainstem and spinal cord and the absence of NPFF-ir fibers in the hypophysis and the olfactory bulbs. This pattern was distinct from that observed for FMRFamide distribution. Striking differences were noted in the pallium, caudal hypothalamus and midbrain tegmentum where FMRFamide-containing cells were localized. The present results in Dermophis support the idea that data from gymnophionans must be included when stating the amphibian condition of a given system because important variations are obvious when gymnophionans are compared with anurans and urodeles.

Topics: Amphibians; Animals; FMRFamide; Immunohistochemistry; Neural Pathways; Oligopeptides; Prosencephalon; Spinal Cord; Tissue Distribution

Neuropeptide FF (NPFF) participates in many physiological functions associated with opioids in the mammalian CNS. We established a pheochromocytoma PC-12 cell line clone stably expressing rat NPFF1 receptors. Both NPFF and FMRFamide activated NPFF1 receptors to couple with Gi/o protein and inhibited adenylyl cyclase activity in PC-12/rNPFF1 cells, but there were no effects on MAPKs (ERK1/2 and p38 MAPK) or PI3K/Akt pathway. FMRFamide also inhibited DARPP-32/Thr34 phosphorylation in the presence of forskolin. Similarly, PFR(Tic)amide, a 'super-agonist' of NPFF receptors, inhibited the production of cAMP and slightly decreased DARPP-32/Thr34 phosphorylation in PC-12/rNPFF1 cells. Intracerebroventricular injections of PFR(Tic)amide blocked behavioral sensitization of locomotor activity to amphetamine, and intrathecal injection of PFR(Tic)amide caused a dose-dependent antinociception in vivo in rats. Thus, "over-activation" of NPFF receptors by PFR(Tic)amide induced different bio-effects from those induced by NPFF in vivo.

Topics: Adenylyl Cyclases; Amphetamine; Analgesia; Animals; Colforsin; Dopamine and cAMP-Regulated Phosphoprotein 32; FMRFamide; Injections, Intraventricular; Injections, Spinal; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Motor Activity; Neuropeptides; Oligopeptides; p38 Mitogen-Activated Protein Kinases; PC12 Cells; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide; Signal Transduction; Tetrahydroisoquinolines

FMRFamide-related peptides (FaRPs) are a large group of neuropeptides containing a common RFamide C-terminus; they have been identified in vertebrates and invertebrates. We have isolated the cDNA that encodes three FaRPs in the tobacco hornworm, Manduca sexta, including the amidated decapeptide F10. The larger FaRPs are the partially processed precursors of F10, a neuropeptide belonging to the myosuppressin family of peptides. The presence of all three FaRPs in different tissues suggests differential utilization of typical dibasic processing sites and atypical processing sites C-terminal to leucine residues. F10 mRNA was detected in the brain, nerve cord, and midgut, and the mRNA levels in the nervous system are dynamically regulated during development. In situ hybridization analysis localized the F10 mRNA to a variety of cell types within the central nervous system (CNS), a peripheral neurosecretory cell (L1), and midgut endocrine cells, which suggests diverse functions. Distribution of the F10-containing neurons within the central nervous system is segment-specific, and the developmental profile suggests that the F10 gene products may have stage-specific functions. Molecular characterization of the F10 gene has provided insights into its regulation and cell-specific distribution that will enhance our understanding of how these FaRPs modulate different physiological systems and ultimately behavior.

Topics: Amino Acid Sequence; Animals; Base Sequence; Brain; Cell Differentiation; DNA, Complementary; Enteric Nervous System; FMRFamide; Ganglia, Invertebrate; Gene Expression Regulation, Developmental; Larva; Manduca; Metamorphosis, Biological; Molecular Sequence Data; Nervous System; Neurons; Neuropeptides; Oligopeptides; RNA, Messenger

An enzymatically stable analog of YGGFMKKKFMRFamide (YFa), a chimeric peptide of metenkephalin and FMRFa, was synthesised. The antinociceptive effects of intracerebroventricular injections of this analog-[D-Ala2)]YAGFMKKKFMRFamide ([D-Ala2]YFa)-was then investigated using the mouse radiant-heat tail-flick test. [D-Ala2]YFa produced modest to good antinociception at 1, 2, and 5 microg/mouse (0.64, 1.28, and 3.22 nmol, respectively). This antinociceptive effect was completely reversed by the opioid receptor antagonist naloxone (1.5 microg/mouse: 4.12 nmol, intracerebroventricular [i.c.v.]), administered 5 min prior. Pretreatment (5 min) with either neuropeptides FF (1 microg/mouse: 0.92 nmol, i.c.v.) or FMRFa (1 microg/mouse: 1.69 nmol, i.c.v.) significantly attenuated the antinociceptive effects induced by [D-Ala2]YFa (1 microg/mouse, i.c.v.). Intracerebroventricular administration of [D-Ala2]YFa at 1 microg/mouse dose with morphine (2 microg/mouse: 5.86 nmol, i.c.v.) produced an additive antinociceptive effect, suggesting that [D-Ala2]YFa may have a modulatory effect on opioid (morphine) analgesia. These results provide further support for a role of such amphiactive sequences in antinociception and its modulation.

Topics: Analgesics, Opioid; Animals; Brain; Drug Interactions; Enkephalin, Methionine; FMRFamide; Injections, Intraventricular; Mice; Mice, Inbred Strains; Morphine; Naloxone; Narcotic Antagonists; Neurons; Nociceptors; Oligopeptides; Pain; Pain Measurement; Recombinant Fusion Proteins

Acidosis is associated with inflammation and ischemia and activates cation channels in sensory neurons. Inflammation also induces expression of FMRFamidelike neuropeptides, which modulate pain. We found that neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe amide) and FMRFamide (Phe-Met-Arg-Phe amide) generated no current on their own but potentiated H+-gated currents from cultured sensory neurons and heterologously expressed ASIC and DRASIC channels. The neuropeptides slowed inactivation and induced sustained currents during acidification. The effects were specific; different channels showed distinct responses to the various peptides. These results suggest that acid-sensing ion channels may integrate multiple extracellular signals to modify sensory perception.

Topics: Acid Sensing Ion Channels; Action Potentials; Animals; Cells, Cultured; Degenerin Sodium Channels; Epithelial Sodium Channels; FMRFamide; Ganglia, Spinal; Humans; Ion Channels; Nerve Tissue Proteins; Neuropeptides; Oligopeptides; Rats; Xenopus

Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HLWAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.

Topics: Amino Acid Sequence; Arrestins; Base Sequence; beta-Arrestins; Calcium; Cell Line; FMRFamide; Humans; Ligands; Molecular Sequence Data; Neuropeptides; Oligopeptides; Receptors, Neuropeptide

FMRFamide and seven FMRFamide-like peptides were manually synthesized on solid phase and their effects tested upon the amplitude of the dorsal root-ventral root monosynaptic reflex (MSR) and dorsal horn field potentials (FP) in an isolated preparation of rat spinal cord. FLFQPQRFamide (NPFF) and AGEGLSSPFWSLAAPQRFamide (NPAF) both depressed a fast component of the FP with similar potencies. FMRFamide also inhibited the FP but its potency was much lower. NPFF and NPAF potentiated the amplitude of the MSR while FMRFamide had no effect. PQRFamide, PFRFamide, FFRFamide, DPQRFamide and Fmoc-FLFQPQRFamide were also examined on the MSR. PQRFamide and PFRFamide potentiated the MSR whereas FFRFamide and DPQRFamide caused a small depression at high concentrations. The increase in amplitude of the MSR induced by NPFF was completely abolished when the N-terminal was left protected with an Fmoc-group. The results suggest that PFRFamide and PQRFamide may act as agonists of NPFF and NPAF whereas the other peptides did not show such activity.

Topics: Amino Acid Sequence; Animals; Dose-Response Relationship, Drug; Electrophysiology; FMRFamide; Ganglia, Spinal; Membrane Potentials; Molecular Sequence Data; Narcotic Antagonists; Oligopeptides; Organ Culture Techniques; Rats; Reflex; Spinal Cord

Mechanical activity was recorded in circular and longitudinal smooth muscle preparations isolated from extensive regions of the porcine gastrointestinal tract in response to the FMRFamide-like neuropeptides F8Famide and A18Famide. In all preparations, the peptides were about equipotent in producing phasic contractions or enhancing spontaneous activity. The most prominent responses were observed in jejunal longitudinal strips which were on the average 91% (+/- 4% SEM, n = 15; 10(-6) M) of the histamine (10(-5) M) responses. The peptide-induced phasic activity was completely abolished by nifedipine but was unaffected by tetrodotoxin, atropine, phentolamine, yohimbine, phenoxybenzamine, propranolol, methysergide, cimetidine, indomethacin, levallorphane or naloxone. Both peptides enhanced acetylcholine-induced contractions. However, bovine ileum and guinea-pig taenia coli was not affected by these peptides. The results indicate that F8F- and A18F-amide contract porcine gastrointestinal smooth muscle by acting directly via non-opioid receptors on L-type calcium channels. In addition an increase of the sensitivity to cholinergic stimulation occurs.

Topics: Animals; Calcium Channels; Calcium Channels, L-Type; Enkephalin, Leucine; Enkephalin, Methionine; FMRFamide; Gastrointestinal Motility; Histamine; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Neuropeptides; Nifedipine; Nitroprusside; Oligopeptides; Swine

FMRFamide is a molluscan peptide that has shown antiopiate activity in a number of mammalian test systems. Peptidomimetics of FMRFamide substituted with conformationally constrained stereoisomers of Z-2,3-methanomethionine or E-2,3-methanomethionine precipitated abstinence syndrome far more potently than FMRFamide itself. The current study determined the effect on antiopiate potency of an additional rigid substitution. A peptidomimetic containing a stereoisomer of E-2,3-methanomethionine was compared with a peptidomimetic additionally substituted at the C-terminal with E-2,3-methanophenylalanine. Morphine abstinence signs were observed after varying doses (0.125-25.0 micrograms) of these two peptidomimetics were injected into the third ventricle of morphine-dependent rats. The peptidomimetic containing both rigid substitutions was far more potent than the peptidomimetic of FMRFamide containing methanomethionine alone. The increased potency appears to be related to enzyme resistance rather than receptor affinity.

Topics: Amino Acid Sequence; Animals; FMRFamide; Invertebrate Hormones; Leucyl Aminopeptidase; Male; Methionine; Molecular Sequence Data; Morphine Dependence; Neuropeptides; Oligopeptides; Opioid Peptides; Phenylalanine; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide; Stereoisomerism

The binding affinities at opioid receptor subtypes in rat or guinea pig brain membranes were determined for the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2), the two mammalian-derived FMRFamide-related peptides F-8-Famide (NPFF; Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe- NH2) and A-18-Famide (Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe -NH2), and the two other FMRFamide-related peptides Tyr-Phe-Met-Arg-Phe-NH2 (Tyr-FMRFamide) and Pro-Gln-Arg-Phe-NH2 (Pro-Gln-RFamide). The mu and delta sites were labeled in rat brain membranes using tritiated [D-Ala2, N-MePhe4,Gly-ol5] enkephalin ([3H]DAMGO) and [D-Ala2,D-Leu5]enkephalin ([3H]DADLE), respectively. The kappa sites were labeled in guinea pig brain using [3H]U-69,593 after treatment with BIT and FIT for kappa 1 and [3H]bremazocine after pretreatment with BIT and FIT for kappa 1 and [3H]bremazocine after pretreatment with BIT and FIT for kappa 2. The kappa 2a binding sites were assayed using [Leu5]enkephalin to block kappa 2b sites and the kappa 2b sites were assayed using (-)-(1S,2S)-U50,488 to block kappa 2a sites. Neither FMRFamide nor any of the FMRFamide-related peptides (up to 61.0 microM) displayed significant affinity at any of the subtypes of opioid receptor. Hence, the known ability of FMRFamide and FaRPs to interact with the opioid system does not appear to be related to direct binding to these opioid receptors.

Topics: Amino Acid Sequence; Animals; FMRFamide; Invertebrate Hormones; Male; Mammals; Molecular Sequence Data; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

The effects of intrathecal injections of F8Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2, 0.05-17.5 nmol) and FMRF-amide (Phe-Met-Arg-Phe-NH2, 0.002-25 nmol), known as anti-opioid agents, were investigated by using noxious thermal (tail flick) and mechanical (paw pressure) tests in the rat. Both peptides produced significant long-lasting (24-48 h) analgesia in both tests without causing detectable motor dysfunction. Pretreatment with systemic naloxone (5.5 mumol/kg i.p.) attenuated the initial antinociceptive effects (first hour) induced by both peptides (8.8 nmol) in the tail flick test and only by FMRFamide in the paw pressure test. A subeffective dose of F8Famide (0.05 nmol) enhanced both the intensity and the duration of spinal morphine (6.6 nmol) analgesia in both tests. In contrast, a subanalgesic dose of FMRFamide (0.002 nmol) decreased the intensity and enhanced the duration of the effect of morphine. These results show that, besides acting as antinociceptive agents in the spinal cord, F8Famide and FMRFamide could differentially modulate spinal opioid functions.

Topics: Amino Acid Sequence; Analgesics; Animals; Dose-Response Relationship, Drug; FMRFamide; Injections, Spinal; Male; Molecular Sequence Data; Morphine; Naloxone; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Pain Measurement; Rats; Rats, Sprague-Dawley; Reaction Time

Neuropeptide FF (FLFQPQRFamide, NPFF) is an octapeptide implicated in morphine analgesia, tolerance and dependence. Many of the behavioral effects of NPFF have also been observed with the invertebrate neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide), which binds to NPFF receptors because of its low homology to the C-terminal portion of NPFF. A competitive ligand binding assay was used to characterize NPFF receptors in rat spinal cord and a strong requirement was found for the C-terminal Arg-Phe-amide. It was found that FMRFamide (Ki = 1.8 nM) bound with lower affinity than NPFF (0.26 nM) but it was about 7-fold more potent than PQRFamide (12 nM). This finding explains the similar bioactivities of NPFF and FMRFamide. The Gln2 appeared to be the cause of the relatively low potency of PQRFamide, based on the binding specificity of NPFF receptors for a series of FMRFamide analogs. In contrast to the Arg-Phe-amide, substitutions at the first and second positions of FMRFamide were generally tolerated, with the most potent analogs being PMRFamide (Ki = 0.54 nM), FFRFamide (0.25 nM) and FWRFamide (0.42 nM). Among the most potent ligands was a pentapeptide containing a photoreactive Phe analog, D-Tyr-(p-benzoyl-Phe)-norLeu-Arg-Phe-amide (Ki = 0.23 nM). It was found that dansyl-PQRFamide and dansyl-RFamide also bound to NPFF receptors with Ki values of 6.1 and 73 nM, respectively. The radioligand binding and G-protein coupling of NPFF receptors were not altered by chronic morphine treatment.

Topics: Amino Acid Sequence; Animals; Central Nervous System; FMRFamide; Male; Molecular Sequence Data; Morphine; Neuropeptides; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide; Spinal Cord; Structure-Activity Relationship

Neuropeptide FF (NFF) is an amidated octapeptide of bovine origin. It has some antiopioid-like effects and it elevates blood pressure when injected intravenously in rats. NFF-immunoreactive nerve cells and terminals are localized in large numbers in the dorsomedial caudal brainstem which is a region involved in central regulation of blood pressure. We compared the localization of NFF-immunoreactive neurons with medullary catecholamine-synthesizing neurons by using immunohistochemical double-labeling and light microscopic mirror methods. NFF and tyrosine hydroxylase coexisted in a minor portion of the NFF neurons in the nucleus of the solitary tract and occasional cell bodies were stained with both NFF and PNMT (phenylethanolamine N-methyltransferase) antisera. The results have anatomical correlation with previous pharmacological reports, suggesting that NFF is present in neuronal systems involved in cardiovascular reflex arcs.

Topics: Animals; Blood Pressure; Catecholamines; FMRFamide; Male; Medulla Oblongata; Nerve Tissue Proteins; Neurons; Neuropeptides; Oligopeptides; Phenylethanolamine N-Methyltransferase; Rats; Rats, Wistar; Tyrosine 3-Monooxygenase

The octapeptide Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F8Fa), originally detected in mammalian brain by antisera raised against the invertebrate peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) is a neuropeptide able to antagonize the actions of both endogenous and exogenous opiates. Since it is well accepted that lymphocytes are targets for opiates, we have tested the effect of F8Fa on T cell proliferation from normal human peripheral blood lymphocytes. Our study shows that F8Fa exerts a concentration-dependent diphasic modulation of human T lymphocyte proliferation. Thus, despite a great variability between individuals, 10(-13) M F8Fa was found to enhance the proliferation of T cells induced by phytohemagglutinin or anti-CD2 monoclonal antibodies, while 10(-7) M F8Fa inhibited T cell proliferation, without affecting cell viability. When F8Fa was tested on monocyte-depleted cell preparations, only the inhibitory effect was observed. These results indicate that F8Fa may stimulate T cells via monocytes, but may also directly inhibit T lymphocyte proliferation. Given the presence of F8Fa-like peptide in human plasma, we suggest that F8Fa may act as a neurohormone in the control of the immune system.

Topics: Carbohydrate Sequence; Cell Division; Dose-Response Relationship, Drug; FMRFamide; Humans; Kinetics; Molecular Sequence Data; Narcotic Antagonists; Neuropeptides; Oligopeptides; Peptides; T-Lymphocytes

Previous studies suggest that neuropeptide FF (NPFF) plays a role in opiate dependence and subsequent abstinence syndrome. The present study assessed the role of NPFF in opiate tolerance. Third ventricular injection of IgG from NPFF antiserum restored the analgesic response to i.c.v. morphine in morphine-tolerant rats (radiant heat tail flick test). IgG from control serum failed to produce this effect. In opiate-naive rats, however, the same treatment with IgG from NPFF antiserum did not affect the analgesic response to i.c.v. morphine. Thus, immunoneutralization of NPFF appears to selectively restore morphine sensitivity in opiate-tolerant animals. These results support the hypothesis that endogenous NPFF contributes to opiate tolerance.

Topics: Amino Acid Sequence; Analgesia; Animals; Cross Reactions; Drug Tolerance; FMRFamide; Immunoglobulin G; Male; Molecular Sequence Data; Morphine; Neuropeptides; Oligopeptides; Pain; Rabbits; Rats; Rats, Inbred Strains

Octapeptide FLFQPQRFamide (FMRFamide-like peptide; morphine-modulating peptide), isolated from bovine brain, has some opiate analgesia modulating effects. Octapeptide FLFQPQRFamide-like immunoreactivity is found in high concentrations in the posterior pituitary, hypothalamus, pons-medulla, and dorsal spinal cord. Octapeptide FLFQPQRFamide-immunoreactive neurons of the brain are localized in the medial hypothalamus and in the nucleus of the solitary tract. High densities of octapeptide FLFQPQRFamide-immunoreactive nerve terminals are found in the median eminence, lateral parabrachial nucleus, and nucleus of the solitary tract. By using the retrograde tract tracing method combined with immunohistochemistry, we studied the central pathways interconnecting the octapeptide FLFQPQRFamide-immunoreactive structures. The octapeptide FLFQPQRFamide-immunoreactive neurons of the hypothalamus sent projections bilaterally to the nucleus of the solitary tract. The octapeptide FLFQPQRFamide-immunoreactive neurons of the nucleus of the solitary tract projected to the contralateral side of the same nucleus, to the lateral parabrachial nuclei bilaterally, and to the ipsilateral periambigual region. The results give neuroanatomical evidence of interacerebral pathways containing recently identified FLFQPQRFamide-like peptides, which may belong to a larger family of peptides. These neuroanatomical findings support the previous pharmacological studies, suggesting that the mammalian FMRFamide-like peptides may, in addition to modulatory effects on nociceptive mechanisms, participate in the regulation of blood pressure, feeding behaviour and endocrine functions.

Topics: Amino Acid Sequence; Animals; Blood Pressure; Brain Chemistry; Brain Mapping; Cattle; Feeding Behavior; FMRFamide; Hypothalamus; Male; Molecular Sequence Data; Neural Pathways; Neurons; Neuropeptides; Oligopeptides; Rats; Rats, Inbred Strains

The effects of two endogenous mammalian FMRFamide (Phe-Met-Arg-Phe-NH2)-related peptides, an octapeptide F8Fa (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) and an octadecapeptide A18Fa (Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe -NH2) on morphine- and restraint stress-induced analgesia and basal nociceptive sensitivity, as measured by the latency of a foot-lifting response to a warmed surface, were examined in male mice. Intracerebroventricular (i.c.v.) administrations of F8Fa and A18Fa (0.10-10 micrograms) during the day-time significantly reduced morphine (10 mg/kg) and restraint-induced analgesia at 30 min after administration, with F8Fa having a greater inhibitory effect than A18Fa. At night during the dark period i.c.v. F8Fa also significantly reduced the elevated nocturnal thermal response latency, while not affecting the shorter day-time nociceptive responses. Peripheral administrations of the prototypic opiate antagonist, naloxone (1.0 mg/kg), had similar inhibitory effects on morphine- and stress-induced analgesia, and the day-night rhythm of nociceptive sensitivity. These results indicate that F8Fa and A18Fa are involved in the modulation of opioid analgesia and suggest that these endogenous FMRFamide-related peptides may be associated with the expression of the day-night rhythm of opioid-mediated nociceptive sensitivity.

Topics: Amino Acid Sequence; Analgesia; Animals; Circadian Rhythm; Endorphins; FMRFamide; Injections, Intraventricular; Male; Mice; Molecular Sequence Data; Morphine; Neuropeptides; Oligopeptides; Pain; Restraint, Physical; Stress, Psychological

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-NH2) is a peptide, originally detected by FMRF-NH2 antisera, and subsequently isolated from bovine brain. Using a specific radioimmunoassay for F-8-F-NH2, we have examined the regional distribution and characteristics of F-8-F-NH2 immunoreactivity (IR) in rat brain, spinal cord and pituitary gland. In CNS, F-8-F-NH2-IR is highly concentrated in the spinal cord, hypothalamus and pons-medulla (368, 202 and 136 fmol per mg protein, respectively); lowest values are in the cortex and hippocampus. A modest rostrocaudal gradient of F-8-F-NH2-IR was observed; levels in the sacral cord are 50% higher than in the cervical cord. Dorsal cord content is 8 times higher than in the ventral cord. Dorsal rhizotomy failed to change F-8-F-NH2-IR in the affected regions of the spinal cord while significantly reducing substance P levels. F-8-F-NH2-IR was significantly decreased caudal to a spinal transection, indicating the presence of a descending pathway within the spinal cord. The highest concentration of F-8-F-NH2-IR (1008 fmol per mg protein) was found in the neurointermediate lobe of the pituitary, while no F-8-F-NH2-IR could be detected in the anterior lobe. Immunohistochemically, F-8-F-NH2-IR was confined to nerve terminal-like structures in the neural lobe. The anterior and intermediate lobes were devoid of immunoreactive structures. HPLC characterization of F-8-F-NH2-IR in the dorsal spinal cord, medulla-pons and pituitary revealed one major immunoreactive peak which is more hydrophobic than bovine F-8-F-NH2. In addition to this material, the hypothalamus was found to contain another, more abundant F-8-F-NH2-immunoreactive peak. Analysis of F-8-F-NH2-IR from posterior pituitary with various antisera having differing affinities for F-8-F-NH2 and gamma 1-MSH indicates that the F-8-F-NH2-IR of rat pituitary is not due to gamma 1-MSH. The high concentration of F-8-F-NH2-like peptide in the dorsal spinal cord supports a role in mediating nociceptive transmission while the localization of F-8-F-NH2-IR in the posterior pituitary suggests an additional autonomic or endocrine function.

Topics: Animals; Brain; FMRFamide; Male; Neuropeptides; Oligopeptides; Pituitary Gland; Radioimmunoassay; Rats; Rats, Inbred Strains; Spinal Cord

Two peptides that are structurally related to the molluscan tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRF-NH2) were recently isolated from bovine brain extract (Yang et al.: Proc. Natl. Acad. Sci. USA 82:7757-7761, '85). These peptides have an attenuating effect on morphine-induced analgesia when injected intracerebroventricularly in rats. Antisera against the two peptides--an octapeptide, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F8F-NH2), and an octadecapeptide, Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe- NH2 (A18F-NH2)--were raised in rabbits and characterized with standard radioimmunoassay and immunohistochemical blocking controls. This study was aimed at localizing neurons in the rat brain that contain immunoreactivity for these peptides. Cryostat sections of normal and colchicine-treated Sprague-Dawley rat brains were incubated with the specific antisera and the immunoreactivity was visualized by the PAP or the FITC method. Immunoreactive neurons were detected in the hypothalamus and nucleus of the solitary tract. Relatively dense networks of fibers and terminals were observed in the lateral parabrachial nucleus and in the nucleus of the solitary tract. Fibers and terminals were also seen in the lateral septum, amygdala, hypothalamus, neurohypophysis, thalamus, periaqueductal gray, and several medullary nuclei. In these areas the immunoreactivity was abolished when the antisera were incubated with the corresponding synthetic peptides F8F-NH2 and A18F-NH2. Preincubation of the antisera with neuropeptide Y (NPY) did not affect the staining. The results suggest that there are neurons in the rat brain that contain F8F-NH2- and A18F-NH2-like peptides. The neuronal system described here may have a role in the regulation of autonomic functions, such as hypothalamic control and respiratory functions. The neuronal connections of the cells containing F8F-NH2- and A18F-NH2-like peptides remain to be studied.

Topics: Amino Acid Sequence; Animals; Brain; Brain Mapping; FMRFamide; Immunohistochemistry; Male; Molecular Sequence Data; Nerve Endings; Neuropeptides; Oligopeptides; Rats; Rats, Inbred Strains

We found that two recently characterized neuropeptides Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2) and Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-NH2 (A-18-F-NH2) elevate mean arterial blood pressure (MAP) in conscious, unrestrained rats. The pressor activities of both agents were attenuated, but not abolished, by prior treatment with guanethidine or prazosin. These results suggest that F-8-F-NH2 and A-18-F-NH2 elevate MAP in rats by potentiating the release of catecholamines and by mechanisms independent of catecholamine release.

Topics: Animals; Blood Pressure; Cross Reactions; Dose-Response Relationship, Drug; FMRFamide; Guanethidine; Immune Sera; Male; Neuropeptides; Oligopeptides; Prazosin; Rats; Rats, Inbred Strains