Lipopolysaccharide induces calcitonin gene-related peptide in the RAW264.7 macrophage cell line.

SUMMARY: Calcitonin gene-related peptide (CGRP) is widely distributed and plays important roles in a wide array of biological functions. It is enriched in primary sensory neurons and hence involved in nociception and neurogenic inflammation. Recent studies have shown that CGRP can be produced by immune cells such as monocytes/macrophages following inflammatory stimulation, suggesting a role in innate immunity. However, it is unclear how CGRP is up-regulated in macrophages and if it plays a role in macrophage functions such as the production of cytokines and chemokines. Using enzyme-linked immunosorbent assay (ELISA) and multiplex ELISA, lipopolysaccharide (LPS) was found to induce CGRP in the RAW 264.7 macrophage cell line. LPS-induced inflammatory mediators such as nerve growth factor (NGF), interleukin-1beta (IL-1beta), IL-6, prostaglandin E(2) (PGE(2)) and nuclear factor-kappaB (NF-kappaB) signalling are involved in inducing CGRP, whereas the NGF receptor trkA and CGRP receptor signalling pathways are unexpectedly involved in suppressing LPS-induced CGRP, which leads to the fine-tune regulation of CGRP release. Exogenous CGRP and CGRP receptor antagonists, in a concentration-dependent manner, stimulated, inhibited or had no effect on basal or LPS-induced release of monocyte chemoattractant protein-1, IL-1beta, IL-6, tumour necrosis factor-alpha and IL-10 in RAW macrophages. The ligand-concentration-dependent regulation of the production of inflammatory mediators by CGRP receptor signalling is a novel mechanism underlying the stimulating and suppressing role of CGRP in immune and inflammatory responses. Together, our data suggest that monocytes/macrophages are an important source of CGRP. Inflammation-induced CGRP has a positive or negative reciprocal effect on the production of other pro- and anti-inflammatory mediators. Thereby CGRP plays both facilitating and suppressing roles in immune and inflammatory responses.

Adrenomedullin-2/intermedin induces cAMP accumulation in dissociated rat spinal cord cells: evidence for the existence of a distinct class of sites of action.

Adrenomedullin-2/intermedin is structurally related to the calcitonin family of peptides, which includes calcitonin gene-related peptide (CGRP), adrenomedullin, and amylin. We recently reported that CGRP and adrenomedullin act through distinct receptors to induce cyclic adenosine monophosphate (cAMP) accumulation in dispersed cells from embryonic rat spinal cord. Here, we investigated the apparent affinity and efficacy of adrenomedullin-2/intermedin for these receptors. Adrenomedullin-2/intermedin competed with [(125)I]-CGRP for binding to specific embryonic spinal cord cells with a pIC(50) of 9.73 +/- 0.06. Interestingly, adrenomedullin-2/intermedin competed for specific [(125)I]-adrenomedullin binding in a biphasic manner with pIC(50) of 9.03 +/- 0.22 and 6.45 +/- 0.24, respectively. Cellular levels of cAMP were increased by adrenomedullin-2/intermedin (pEC(50) 7.84 +/- 0.08) when cells were exposed to this peptide for 10 min at 37 degrees C. This effect was partially inhibited by the non-peptide antagonist BIBN4096BS (pA(2) 6.56 +/- 0.12), the adrenomedullin antagonist hAM(22-52) (pA(2) 6.36 +/- 0.30), and the adrenomedullin/CGRP antagonist CGRP(8-37) (pA(2) 7.24 +/- 0.60). More interestingly, a highly significant effect of adrenomedullin-2/intermedin on cAMP accumulation (pEC(50) 7.3 +/- 0.14) was still observed even in the presence of a mixture of saturating concentrations of BIBN4096BS, hAM(22-52), and the amylin antagonist AC187. Taken together, these data provide evidence for the possible existence of a distinct class of receptor sites for adrenomedullin-2/intermedin in embryonic rat spinal cord cells.

Distribution of peripherally injected peptide YY ([125I] PYY (3-36)) and pancreatic polypeptide ([125I] hPP) in the CNS: enrichment in the area postrema.

The mechanism by which blood-borne peptide YY (3-36) (PYY(3-36)) and pancreatic polypeptide (PP) inhibit food intake is not clear and could implicate peripheral (vagal afferent pathways) and/or central (direct action on specific brain nuclei) mechanisms. To identify the primary brain structure(s) that could be activated after a peripheral injection of neuropeptide Y-related peptides, we investigated the distribution of radioactive materials using whole body autoradiography and coronal brain sections. Rats were injected with [125I] porcine (p) PYY(3-36) (i.p., 10 microCi) and killed after 30 min, 1, 2, or 4 h. After i.p. administration, significant amounts of radioactive materials were rapidly (<30 min) detected in the blood circulation and various tissues including the kidneys, liver, lung, heart, bone marrow, gastrointestinal tract, and thyroid gland, whereas in the brain, low but significant amounts of radioactive materials were detected at the level of the area postrema. Next, we investigated the distribution of radioactive labeling in the brain after i.v. injections of [125I]pPYY(3-36) (Y2 and Y5 subtypes), [125I] human (h) PP (Y4 and Y5 receptors), and [125I][Leu(31), Pro(34)] pPYY (Y1, Y4 and Y5 classes) in the rat brain. Fifteen minutes post injection, autoradiograms revealed positive signals only in the area postrema after the injection of [125I]-hPP and [125I][Leu(31), Pro(34)]pPYY. Whereas the presence of [125I]pPYY(3-36)-related labeling was detected in the area postrema, subfornical organ, and median eminence. In all other brain structures, including all hypothalamic nuclei and other circumventricular organs, near background level signals were detected. These data suggest that the inhibition of food intake observed after peripheral injections of pPYY(3-36) and hPP could involve receptor activation preferentially located at the level of the area postrema, a structure well-known to be involved in the modulation of food intake.

Dcc haploinsufficiency regulates dopamine receptor expression across postnatal lifespan.

Adolescence is a period during which the medial prefrontal cortex (mPFC) undergoes significant remodeling. The netrin-1 receptor, deleted in colorectal cancer (DCC), controls the extent and organization of mPFC dopamine connectivity during adolescence and in turn directs mPFC functional and structural maturation. Dcc haploinsufficiency leads to increased mPFC dopamine input, which causes improved cognitive processing and resilience to behavioral effects of stimulant drugs of abuse. Here we examine the effects of Dcc haploinsufficiency on the dynamic expression of dopamine receptors in forebrain targets of C57BL6 mice. We conducted quantitative receptor autoradiography experiments with [3H]SCH-23390 or [3H]raclopride to characterize D1 and D2 receptor expression in mPFC and striatal regions in male Dcc haploinsufficient and wild-type mice. We generated autoradiograms at early adolescence (PND21±1), mid-adolescence (PND35±2), and adulthood (PND75±15). C57BL6 mice exhibit overexpression and pruning of D1, but not D2, receptors in striatal regions, and a lack of dopamine receptor pruning in the mPFC. We observed age- and region-specific differences in D1 and D2 receptor density between Dcc haploinsufficient and wild-type mice. Notably, neither group shows the typical pattern of mPFC dopamine receptor pruning in adolescence, but adult haploinsufficient mice show increased D2 receptor density in the mPFC. These results show that DCC receptors contribute to the dynamic refinement of D1 and D2 receptor expression in striatal regions across adolescence. The age-dependent expression of dopamine receptor in C57BL6 mice shows marked differences from previous characterizations in rats.

Emotional behavior in aged neuropeptide Y (NPY) Y2 knockout mice.

Neuropeptide Y (NPY) was shown to modulate anxiety- and depression-related behaviors in various animal models. Previous studies demonstrated that NPY Y2 receptor knockout (KO) mice display an anxiolytic- and antidepressant-like phenotype compared with control animals. However, the long-term effect of the deletion of this receptor in aged animals is unknown. Thus, anxiety- and depression-related behaviors were investigated in 2-yr-old NPY Y2 KO mice. Aged NPY Y2 KO mice display an anxiolytic-like profile as assessed in the elevated plus-maze and open field, providing further support for a role for Y2 receptors in anxiety-related behaviors. Furthermore, aged NPY Y2 KO mice have significantly lower immobility scores in the forced swim test; supporting the role for this receptor in antidepressant-like behaviors. These data provide further evidence that modulators of the NPY Y2 receptor subtype are drug targets for the treatment of anxiety and mood disorders in human subjects.

Selective mediation of nerve injury-induced tactile hypersensitivity by neuropeptide Y.

Prevention of nerve injury-induced tactile, but not thermal, hypersensitivity is achieved by ipsilateral lesions of the dorsal columns or lidocaine microinjection into the nucleus gracilis (n. gracilis). These and other data support the possibility that tactile hyperresponsiveness after nerve injury may be selectively mediated by a low-threshold myelinated fiber pathway to the n. gracilis. Here we identify a transmitter that might selectively mediate such injury-induced tactile hypersensitivity. Neuropeptide Y (NPY), normally not detected in the dorsal root ganglion (DRG) or in the n. gracilis of rats, became markedly upregulated at both sites and in the spinal cord after spinal nerve injury. Injury-induced NPY-IR occurred predominately in large-diameter DRG cells, and the NPY-IR in the n. gracilis was blocked by dorsal rhizotomy or dorsal column lesion. NPY microinjection into the n. gracilis of uninjured rats elicited reversible tactile, but not thermal, hypersensitivity only in the ipsilateral hindpaw. Administration of anti-NPY antiserum, but not control serum or preabsorbed serum, into the n. gracilis ipsilateral to nerve injury reversed tactile, but not thermal, hypersensitivity. Similarly, microinjection of the NPY antagonists NPY(18-36) and (R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphenylacetyl)-argininamide trifluoroacetate, into the n. gracilis ipsilateral to the injury reversed tactile, but not thermal, hypersensitivity. Antagonist administration into the contralateral n. gracilis had no effect on injury-induced hypersensitivity. These data suggest the selective mediation of nerve injury-induced tactile hypersensitivity by upregulated NPY via large fiber input to n. gracilis. Selective reversal of injury-induced tactile allodynia by NPY receptor antagonists would have significant implications for human neuropathic conditions.

BODIPY-conjugated neuropeptide Y ligands: new fluorescent tools to tag Y1, Y2, Y4 and Y5 receptor subtypes.

N-terminal labelled fluorescent BODIPY-NPY peptide analogues were tested in Y1, Y2, Y4 and Y5 receptor-binding assays performed in rat brain membrane preparations and HEK293 cells expressing the rat Y1, Y2, Y4 and Y5 receptors. BODIPY TMR/FL-[Leu31, Pro34]NPY/PYY were able to compete for specific [125][Leu31, Pro34]PYY-binding sites with an affinity similar to that observed for the native peptide at the Y1 (Ki=1-6 nM), Y2 (Ki>1000 nM), Y4 (Ki=10 nM) and Y5 (Ki=1-4 nM) receptor subtypes. BODIPY FL-PYY(3-36) was able to compete for specific Y2 (Ki=10 nM) and Y5 (Ki=30 nM) binding sites, but had almost no affinity in Y1 and Y4 assays. BODIPY FL-hPP was able to compete with high affinity (Ki; 1 and 15 nM) only in Y4 and Y5 receptor-binding assays. BODIPY TMR-[cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP and BODIPY TMR-[hPP(1-17), Ala31, Aib32]NPY were potent competitors only on specific Y5-binding sites (Ki=0.1-0.6 nM). As expected, these fluorescent peptides inhibited forskolin-induced cAMP accumulation, demonstrating that they retained their agonist properties. When tested in confocal microscopy imaging, fluorescent Y1 and Y5 agonists internalized in a time-dependent manner in Y1 and Y5 transfected cells, respectively. These results demonstrate that BODIPY-conjugated NPY analogues retain their selectivity, affinity and agonist properties for the Y1, Y2, Y4 and Y5 receptor subtypes, respectively. Thus, they represent novel tools to study and visualize NPY receptors in living cells.

Role of serotonin (5-HT) in the antidepressant-like properties of neuropeptide Y (NPY) in the mouse forced swim test.

Neuropeptide Y (NPY) is thought to be implicated in depressive disorders. The mouse forced swim test (FST) is an animal model widely used as a predictor of the efficacy of antidepressant drugs. The present study was undertaken to explore the possible contribution of endogenous serotonin (5-HT) systems in the behavioral effects elicited by NPY in this model. The selective serotonin re-uptake inhibitor (SSRI), fluoxetine, was also tested for comparison. 5-HT was depleted prior to testing by the administration of the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA; 300 mg/kg, i.p., each day for 3 days; control mice received saline-vehicle over the same period). On the fourth day, mice received NPY (3 nmol, I.C.V.), fluoxetine (16 mg/kg, i.p.) or saline injections before testing in the FST. Both NPY and fluoxetine significantly reduced immobility time in saline-treated control animals. Pre-treatment with PCPA significantly blocked the effects of fluoxetine in the FST, confirming the role of endogenous 5-HT. Similarly, pre-treatment with PCPA also significantly attenuated the anti-immobility effects of NPY, thus suggesting a role for 5-HT in the effects of NPY in the FST. Quantitative receptor autoradiography revealed increases in specific [125I][Leu31, Pro34]PYY sites that were sensitive to BIBP3226 (Y1-like sites) in various brain regions. Specific [125I]GR231118 and [125I]PYY(3-36) binding levels were not changed following PCPA treatment, suggesting that depletion of endogenous 5-HT resulted in an apparent increase in the level of Y1 sites in their high-affinity state. Taken together, these results suggest a role for 5-HT-related systems in the antidepressant-like properties of NPY.

Immunohistochemical distribution of neuropeptide Y, peptide YY, pancreatic polypeptide-like immunoreactivity and their receptors in the epidermal skin of healthy women.

Few studies have suggested that neuropeptide Y (NPY) could play an important role in skin functions. However, the expression of NPY, the related peptides, peptide YY (PYY) and pancreatic polypeptide (PP) and their receptors have not been investigated in human skin. Using specific antisera directed against NPY, PYY, PP and the Y1, Y2, Y4 and Y5 receptor subtypes, we investigated here the expression of these markers. NPY-like immunoreactivity (ir) in the epidermal skin could not be detected. For the first time we report the presence of positive PP-like ir immunofluorescent signals in epidermal cells, i.e. keratinocytes of skin from three areas (abdomen, breast and face) obtained as surgical left-overs. The immunofluorescent signal of PP-like ir varies from very low to high level in all three areas. In contrast, PYY-like ir is only expressed in some cells and with varied level of intensity. Furthermore and for the first time we observed specific Y1 and Y4 receptor-like ir in all epidermal layers, while the Y2 and Y5 subtypes were absent. Interestingly, as seen in human epidermis, in Episkin, a reconstituted human epidermal layer, we detected the presence of PP-like as well as Y1-like and Y4-like ir. These data have shown the presence and distribution of PYY, PP and Y1 and Y4 receptors in the human skin and Episkin, suggesting possible novel roles of NPY related peptides and their receptors in skin homeostasis.

The neurocircuitry and receptor subtypes mediating anxiolytic-like effects of neuropeptide Y.

This review aims to give a brief overview of NPY receptor distribution and physiology in the brain and summarizes series of studies, test by test and region by region, aimed at identification receptor subtypes and neuronal circuitry mediating anxiolytic-like effects of NPY. We conclude that from four known NPY receptor subtypes in the rat (Y(1), Y(2), Y(4), Y(5)), only the NPY Y(1) receptor can be linked to anxiety-regulation with certainty in the forebrain, and that NPY Y(2) receptor may have a role in the pons. Microinjection studies with NPY and NPY receptor antagonists support the hypothesis that the amygdala, the dorsal periaqueductal gray matter, dorsocaudal lateral septum and locus coeruleus form a neuroanatomical substrate that mediates anxiolytic-like effects of NPY. The release of NPY in these areas is likely phasic, as NPY receptor antagonists are silent on their own. However, constant NPY-ergic tone seems to exist in the dorsal periaqueductal gray, the only brain region where NPY Y(1) receptor antagonists had anxiogenic-like effects. We conclude that endogenous NPY has an important role in reducing anxiety and serves as a physiological stabilizer of neural activity in circuits involved in the regulation of arousal and anxiety.

The neuropeptide Y (NPY) Y1 receptor subtype mediates NPY-induced antidepressant-like activity in the mouse forced swimming test.

The present study was undertaken to investigate the possible antidepressant-like effects of neuropeptide Y (NPY) in the mouse forced swimming test, an animal model widely used for the screening of potential antidepressant drugs. In addition, experiments were performed, using agonists and selective antagonists, to assess the potential role of NPY Y(1) and Y(2) receptor subtypes in this model. Complementary studies were performed in an open field apparatus to rule out any changes in locomotor activity that might have interfered with the interpretation of data from the mouse forced swimming test. Intracerebroventricular injections (0.03 nmole-3 nmole) of NPY, [Leu(31)Pro(34)]PYY (Y(1) agonist), NPY(13-36) (Y(2) agonist), BIBP3226, BIBO3304 (Y(1) antagonists) and BIIE0246 (Y(2) antagonist) were performed 30 min prior to testing in the mouse forced swimming test and open field. NPY administration significantly reduced immobility time in a dose dependent manner (p <.01 vs. control group), as did [Leu(31)Pro(34)]PYY (p <.01 vs. control group) and BIIE0246 (p <.05 vs. control group). In contrast, BIBO3304, BIBP3226 and NPY(13-36) did not display any activity at the doses tested. However, pretreatment with BIBO3304 or BIBP3226 significantly blocked the anti-immobility effects of NPY. Data from the open field demonstrated that BIIE0246 increased horizontal ambulation at the dose found to be active in the forced swimming test. Taken together, our results demonstrate that NPY displays antidepressant-like activity in the mouse forced swimming test, and suggest that this activity is mediated by the NPY Y(1) receptor subtype.

Development and characterization of a highly selective neuropeptide Y Y5 receptor agonist radioligand: [125I][hPP1-17, Ala31, Aib32]NPY.

(1) The existence of multiple classes of neuropeptide Y (NPY) receptors (Y(1), Y(2), Y(4), Y(5) and y(6)) is now well established. However, one of the major difficulties in the study of these various receptor subtypes is the current lack of highly selective probes to investigate a single receptor class. Up to most recently, this was particularly true for the Y(4) and Y(5) subtypes. (2) [hPP(1-17), Ala(31), Aib(32)]NPY, the first highly selective Y(5) agonist, was iodinated using the chloramine T method and purified by high-pressure liquid chromatography. (3) Binding performed in rat brain homogenates revealed that equilibrium was reached after 120 min (t(1/2)=21 min) and 60 min (t(1/2)=12 min) at 25 and 100 pM [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY, respectively. (4) Isotherm saturation binding experiments demonstrated that [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY binds to an apparent single population with high-affinity (K(D) of 1.2 and 1.7 nM) and low-capacity (B(max) of 14+/-3 fmol/100,000 cells and 20+/-5 fmol/mg protein) sites in Y(5) receptor HEK293-transfected cells and rat brain membrane homogenates, respectively. No specific [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY binding sites could be detected in Y(1), Y(2) or Y(4) receptors transfected HEK293 cells, demonstrating the high selectivity of this ligand for the Y(5) subtype. (5) Competition binding experiments performed in rat brain membrane homogenates and Y(5)-receptor transfected HEK293 cells demonstrated that specific [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY binding was competed with high affinity by Y(5) agonists and antagonists such as [Ala(31), Aib(32)]NPY, [hPP(1-17), Ala(31), Aib(32)]NPY, hPP, CGP71683A and JCF109, but not by Y(1) (BIBP3226), Y(2) (BIIE0246) and Y(1)/Y(4) (GR231118) preferential ligands. (6) Taken together, these data demonstrate that [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY is the first highly selective Y(5) radioligand to be developed. This new probe should prove most useful for further detailed studies of the molecular and pharmacological properties of this receptor subtype in brain and peripheral tissues.

Agonist- and antagonist-induced sequestration/internalization of neuropeptide Y Y1 receptors in HEK293 cells.

1 Neuropeptide Y Y(1) receptors are known to internalize following the binding of agonists. In the present study, a pseudopeptide Y(1) receptor antagonist, homodimeric Ile-Glu-Pro-Dpr-Tyr-Arg-Leu-Arg-Tyr-CONH(2) (GR231118), also induced Y(1) receptor internalization in human embryonic kidney (HEK293) cells. 2 We demonstrated first that both specifically bound radiolabeled antagonist ([(125)I]GR231118) and agonist ([(125)I][Leu(31), Pro(34)]PYY) underwent receptor-mediated sequestration/internalization in transfected HEK293 cells. 3 Agonist-induced Y(1) receptor internalization was dependent on clathrin-coated pits and was regulated in part by Gi/o-protein activation as revealed by pertussin toxin sensitivity. In contrast, antagonist-induced sequestration of Y(1) receptors was partly dependent on clathrin-coated pits, but independent from Gi/o-protein activation. 4 Exposure to high concentrations of agonist or antagonist caused a 50 and 75% loss of cell surface binding, respectively. The loss caused by the agonist rapidly recovered. This phenomenon was blocked by monensin, an inhibitor of endosome acidification, suggesting that cell surface receptor recovery is due to recycling. In contrast to the agonist, GR231118 induced a long-lasting sequestration of Y(1) receptors in HEK293 cells. 5 Immunofluorescence labeling indicated that following 40 min of incubation with either the agonist or the antagonist, Y(1) receptors followed markedly different intercellular trafficking pathways. 6 Taken together, these findings provided evidence that a pseudopeptide Y(1) receptor antagonist can induce long-lasting disappearance of cell surface receptors through a pathway distinct from the classical endocytic/recycling pathway followed by stimulation with an agonist.

Characterization of neuropeptide Y, Y(2) receptor knockout mice in two animal models of learning and memory processing.

Neuropeptide Y (NPY) and, in particular, the Y2 receptor subtype, has been suggested to be involved in learning and memory processing. However, the precise role of Y2 receptors in learning and memory remains unclear. In the present study, mice lacking NPY Y2-type receptors were assessed in two animal models of learning and memory processing. We found that NPY Y2-/- mice displayed a deficit on the probe trial in the Morris water maze task, whereas acquisition performance, swim speed, and visible platform performance did not differ significantly between groups. In addition, NPY Y2-/- mice exhibited a marked deterioration in object memory 6 h, but not 1 h, following initial exposure in the object recognition test. Both groups of mice showed similar locomotor activity profiles in a low-stress, open field test. These data support the hypothesis that Y2 receptors are involved in the regulation of learning and memory processing.

Receptor autoradiography as mean to explore the possible functional relevance of neuropeptides: focus on new agonists and antagonists to study natriuretic peptides, neuropeptide Y and calcitonin gene-related peptides.

Over the past 20 years, receptor autoradiography has proven most useful to provide clues as to the role of various families of peptides expressed in the brain. Early on, we used this method to investigate the possible roles of various brain peptides. Natriuretic peptide (NP), neuropeptide Y (NPY) and calcitonin (CT) peptide families are widely distributed in the peripheral and central nervous system and induced multiple biological effects by activating plasma membrane receptor proteins. The NP family includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The NPY family is composed of at least three peptides NPY, peptide YY (PYY) and the pancreatic polypeptides (PPs). The CT family includes CT, calcitonin gene-related peptide (CGRP), amylin (AMY), adrenomedullin (AM) and two newly isolated peptides, intermedin and calcitonin receptor-stimulating peptide (CRSP). Using quantitative receptor autoradiography as well as selective agonists and antagonists for each peptide family, in vivo and in vitro assays revealed complex pharmacological responses and radioligand binding profile. The existence of heterogeneous populations of NP, NPY and CT/CGRP receptors has been confirmed by cloning. Three NP receptors have been cloned. One is a single-transmembrane clearance receptor (NPR-C) while the other two known as CG-A (or NPR-A) and CG-B (or NPR-B) are coupled to guanylate cyclase. Five NPY receptors have been cloned designated as Y(1), Y(2), Y(4), Y(5) and y(6). All NPY receptors belong to the seven-transmembrane G-protein coupled receptors family (GPCRs; subfamily type I). CGRP, AMY and AM receptors are complexes which include a GPCR (the CT receptor or CTR and calcitonin receptor-like receptor or CRLR) and a single-transmembrane domain protein known as receptor-activity-modifying-proteins (RAMPs) as well as an intracellular protein named receptor-component-protein (RCP). We review here tools that are currently available in order to target each NP, NPY and CT/CGRP receptor subtype and establish their respective pathophysiological relevance.

Characterization of a new neuropeptide Y Y5 agonist radioligand: [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP.

In order to optimally characterize a class of neuropeptide Y (NPY) receptors expressed in a tissue enriched with multiple subtypes (Y1, Y2, Y4 and Y5) and to establish its detailed distribution, it is critical to use highly selective and specific probes that possess very low non-specific binding. In that context, we recently reported on the development of [125I][hPP(1-17), Ala31, Aib32]NPY as Y5 receptor radioligand. However, the non-specific binding obtained with this radioligand was too high to allow for detailed receptor autoradiography studies [Br. J. Pharmacol. 139 (2003) 1360]. Iodinated [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP may represent a better Y5 radioligand in that regard. Accordingly, [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding was investigated in rat brain membrane homogenates and its specificity and selectivity established in rat Y1, Y2, Y4 and Y5 transfected HEK293 cells. No specific binding was detected in HEK293 cells transfected with the rat Y1, Y2 or Y4 receptors, while saturable binding was observed in cells transfected with the rat Y5 receptor cDNA and in rat brain membrane homogenates (KD of 0.5-0.7 nM). Competition binding experiments performed in rat brain membrane homogenates demonstrated that specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding was competed with nanomolar affinities by Y5 agonists and antagonists such as [Leu31,Pro34]PYY, PYY(3-36), [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP, [Ala31, Aib32]NPY, [hPP(1-17), Ala31, Aib32]NPY, CGP71683A and JCF109, but not by Y1 (BIBP3226 and BIBO3304), Y2 (BIIE0246) and Y4 (GR231118) ligands. Non-specific binding was also lower than that reported for [125I][hPP(1-17), Ala31, Aib32]NPY. Interestingly, detailed analysis of competition binding curves obtained with [Leu31, Pro34]PYY, hPP, PYY(3-36) and [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP against specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP sites were best fitted to a two-site model. Additionally, receptor autoradiography studies revealed the presence of specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding sites in the lateral septum and area postrema while other brain regions contained much lower levels of specific binding. Taken together, these data suggest that [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP represents a useful tool to study the unique feature of the Y5 receptor subtype.

Neuropeptide Y (NPY) Y2 receptors mediate behaviour in two animal models of anxiety: evidence from Y2 receptor knockout mice.

The behavioural phenotype of mice lacking neuropeptide Y (NPY) Y(2)-type receptors was assessed in two well documented animal models of anxiety: namely, the elevated plus maze and the open field. NPY Y(2)-/- mice made more entries into, and spent significantly more time on, the open arms of the elevated plus maze when compared to their wild-type Y(2)+/+ controls (P<0.001). This effect was not due to non-specific changes in locomotor activity as the number of closed arm entries did not differ between groups. In addition, NPY Y(2)-/- mice displayed increased preference for the central area of the open field when compared to Y(2)+/+ animals (P<0.01), whereas total entries did not differ between groups. This study suggests that NPY Y(2) receptors may play an inhibitory role and supports the hypothesis that Y(2) receptors are involved in the regulation of anxiety-like behaviours by NPY.

Aged neuropeptide Y transgenic rats are resistant to acute stress but maintain spatial and non-spatial learning.

The behavioral phenotype of five-month-old rats overexpressing neuropeptide Y (NPY) has previously been described [Proc Natl Acad Sci USA 97 (2000) 12852]. In this transgenic rat model, there is central overexpression of prepro-NPY mRNA and NPY peptide in the hippocampus and hypothalamus and decreased Y1 binding sites within the hippocampus. These molecular and neurochemical events led to altered anxiety profile and learning abilities in NPY-overexpressing rats. In the present study, anxiety and learning/memory related behaviors were examined in one-year-old NPY-transgenic rats in order to assess any behavioral changes that may have occurred during the aging process. As observed in 5-month-old overexpressing rats, aged NPY-transgenic animals are resistant to acute physical restraint stress measured by the elevated-plus maze and demonstrate anxiolytic-like activity in the open field. However, in contrast to data in young rats, there was no significant difference between aged wildtype and NPY-transgenic animals in relation to spatial and non-spatial memory as indicated by the (allo- and ego-centric) Morris water maze and object recognition test. It would thus appear that the anxiolytic-like profile observed in young NPY-overexpressing rats is maintained in older animals providing further evidence for a role for NPY in anxious behaviors. However, the cognitive deficits observed in young rats do not appear to occur in older animals suggesting the existence of compensatory mechanisms leading to a reversal of the learning deficits noted in younger animals. These results also provide additional evidence for the mechanistic dissociation between anxiety and cognition-related behaviors modulated by NPY.

Adrenomedullin receptor binding sites in rat brain and peripheral tissues.

The existence of specific adrenomedullin receptor binding sites was investigated using the agonist peptide fragment [125I]human adrenomedullin-(13-52) in rat brain, lung and vas deferens homogenates. Saturation-binding experiments suggest that [125I]human adrenomedullin-(13-52) binds to an apparent single population of sites with similar affinities (K(D) of 0.3 to 0.6 nM) but with different maximal binding capacity in the rat brain, lung and vas deferens homogenates (B(max) of 73, 1760 and 144 fmol/mg protein, respectively). Competition-binding experiments using various analogues and fragments of calcitonin gene-related peptide (CGRP) and adrenomedullin were also performed using this radioligand. Competition-binding profiles suggest the possible existence of heterogeneous populations of adrenomedullin receptor binding sites. For example, in rat brain, human adrenomedullin-(1-52) and human adrenomedullin-(13-52) competed against specific [125I]human adrenomedullin-(13-52) sites with competition curves best fitted to a two-site model. Additionally, human calcitonin gene-related peptide alpha (hCGRPalpha), [Cys(Et)(2,7)]hCGRPalpha and [[R-(R,(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide] (BIBN4096BS) competed against specific [125I]human adrenomedullin-(13-52) binding with profiles that were also best fitted to a two-site model. Furthermore, binding assays performed in the presence of GTPgammaS (100 microM) revealed that this compound inhibited 20% of specific [125I]human adrenomedullin-(13-52) sites in rat brain homogenates and competition curves of human adrenomedullin-(1-52) and [Cys(Et)(2,7)]hCGRPalpha against specific [125I]human adrenomedullin-(13-52) sites remained best fitted to a two-site model. Moreover, the existence of specific [125I]human adrenomedullin-(13-52) binding sites that are resistant to human adrenomedullin-(22-52) and human CGRP-(8-37) is suggested in the rat brain and vas deferens. Taken together, these data provide evidence for the possible existence of heterogeneous populations of adrenomedullin binding sites in rat brain and peripheral tissues.

Pharmacological characterization of human NPFF(1) and NPFF(2) receptors expressed in CHO cells by using NPY Y(1) receptor antagonists.

Neuropeptide FF (NPFF) belongs to an opioid-modulatory system including two precursors (pro-NPFF(A) and pro-NPFF(B)) and two G-protein coupled receptors (NPFF(1) and NPFF(2)). The pharmacological and functional profiles of human NPFF(1) and NPFF(2) receptors expressed in Chinese hamster ovary (CHO) cells were compared by determining the affinity of several peptides derived from both NPFF precursors and by measuring their abilities to inhibit forskolin-induced cAMP accumulation. Each NPFF receptor recognizes peptides from both precursors with nanomolar affinities, however, with a slight preference of pro-NPFF(A) peptides for NPFF(2) receptors and of pro-NPFF(B) peptides for NPFF(1) receptors. BIBP3226 ((R)-N(2)-(diphenylacetyl)-N-[(4-hydroxyphenyl)-methyl]-argininamide) and BIBO3304 ((R)-N(2)-(diphenylacetyl)-N-[4-(aminocarbonylaminomethyl)-benzyl]-argininamide trifluoroacetate), two selective neuropeptide Y (NPY) Y(1) receptor antagonists, display relative high affinities for NPFF receptors and exhibit antagonist properties towards hNPFF(1) receptors. The structural determinants responsible for binding of these molecules to NPFF receptors were investigated and led to the synthesis of hNPFF(1) receptor antagonists with affinities from 40 to 80 nM. Our results demonstrate differences in pharmacological characteristics between NPFF(1) and NPFF(2) receptors and the feasibility of subtype-selective antagonists.