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NEUROPHARMACOLOGY
Douglas Hospital Research Centre, Department of Psychiatry, McGill University, Montreal, Québec, Canada (Y.D., R.Q.); Laboratoire de Physiologie Neurovégétative, Unité Mixte de Recherche Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique-Université, Aix-Marseille III, Faculté St-Jérôme, Marseille, France (E.M.); and Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Montréal, Québec, Canada (A.F.)
Received for publication
May 10, 2005
Accepted
June 7, 2005.
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Abstract |
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). NPY and its related peptides have been implicated in several physiological functions, including regulation by the central nervous system of feeding, cardiorespiratory parameters, and anxiety-related behaviors (Vezzani et al., 1999; Kask et al., 2002; Berglund et al., 2003a; Kalra and Kalra, 2004). The various effects of this peptide family are mediated by the activation of at least five receptor subtypes designated as Y1, Y2, Y4, Y5, and y6 (Michel et al., 1998). They have all been cloned and belong to the seven transmembrane G protein-coupled receptor type 1 superfamily (Michel et al., 1998; Dumont et al., 2004a). They are expressed as functional receptors in various rat and human tissues, except for the y6 subtype, which is restricted to mouse and rabbit (Michel et al., 1998).
Various studies reported on the discrete neuroanatomical distribution of NPY receptor subtypes. For example, using 125I-[Leu31,Pro34]hPYY as radioligand in the presence of selective nonpeptide Y1 receptor antagonists such as BIBP3226 and BIBO3304, we proposed that residual specific labeling had a ligand selectivity profile very similar to that of the Y5 receptor subtype in the rat CNS (Dumont et al., 1998). These results were confirmed using the Y5 receptor antagonist CGP71683A (Dumont et al., 2000a). However, under these assay conditions, the possible recognition of the Y4 subtype could not be fully excluded because [Leu31,Pro34]PYY is known to possess nanomolar affinity for this receptor (Gehlert et al., 1996). It is interesting that, using purported Y4 ligands such as 125I-hPP, 125I-rPP, and 125I-labeled bovine PP, we were able to visualize a very discrete distribution of specific receptor sites with moderate to very high amounts seen in a few regions, including the medial preoptic area, paraventricular nucleus of the hypothalamus, and interpeduncular nucleus (Trinh et al., 1996; Gehlert et al., 1997). Furthermore, quantitative receptor autoradiography has demonstrated that labeling seen with 125I-rPP (a Y4 ligand with low affinity for the Y5 receptor) was not identical to that of specific 125I-hPP. In some rat brain nuclei such as the interpeduncular nuclei, nucleus tractus solitarius, and area postrema, higher levels of specific 125I-hPP binding sites were detected compared with specific 125I-rPP binding (Trinh et al., 1996), suggesting that 125I-hPP, in addition to Y4 receptors, recognized another population of sites, most likely the Y5 subtype. However, some brain structures labeled by 125I-PPs failed to be enriched with specific 125I-[Leu31,Pro34]PYY (Gehlert et al., 1997) and 125I-GR231118 (Dumont and Quirion, 2000; Schober et al., 2000) binding sites even if these two radioligands are known to possess high affinities for the Y4 receptor subtype (Dumont et al., 2004a). These results could be taken as evidence for the existence of yet another class of sites preferentially recognized by PP-related molecules. Moreover and rather surprisingly, using a novel highly selective Y5 radioligand, 125I-[cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP, we could not detect specific binding in the nucleus tractus solitarius (Dumont et al., 2004b), contrasting with our previous reports on the distribution of the Y5-like receptor subtype in rat brain using 125I-[Leu31,Pro34]hPYY under Y1 masking conditions (Dumont et al., 1998, 2000a). In other regions, such as the area postrema, specific 125I-[cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP, and 125I-[Leu31,Pro34]hPYY/Y1-insensitive binding sites were similarly distributed (Dumont et al., 1998, 2004b).
To clarify these apparent discrepancies, we investigated here the ligand selectivity profile and discrete distribution of specific 125I-[Leu31,Pro34]hPYY and 125I-hPP binding sites using a variety of Y1, Y4, and Y5 related molecules as competitors or masking agents. Our results suggest the existence of additional subtype(s) of receptor binding sites for NPY and related peptides in the rat brain.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). The Y5 agonists [Ala31,Aib32]pNPY, [hPP(1-17),Ala31,Aib32]hNPY, and [cPP(1-7), NPY(19-23),Ala31,Aib32,Gln34]hPP were gifts from Dr. Annette Beck Sickinger (University of Leipzig, Leipzig, Germany), whereas rPP was purchased from Bachem California (Torrance, CA). BIBP3226, BIBO3304, and BIIE0246 were generously provided by Boehringer Ingelheim GmbH (Ingelheim, Germany), whereas GR231118 was a gift from GlaxoSmithKline (Research Triangle Park, NC). CP732925, CP760542, and CP781214 were generously provided by Pfizer, Inc. (Groton, CT). JCF109 and CGP71683A were graciously obtained from Servier (Paris, France). Bovine serum albumin (BSA) and iodine-125 were obtained from Valeant Pharmaceuticals Canada Ltd. (Montréal, QC, Canada), and bacitracin was purchased from Sigma Chemical (St. Louis, MO). Whatman Schleicher and Schuell #32 glass filters were obtained from VWR-Canlab (Montréal, QC, Canada). All other chemicals were of analytical grade and obtained from Fisher Scientific (Montreal, QC, Canada) or Sigma Chemical.
Iodine-125 was incorporated into the tyrosine residue of hPP, [Leu31,Pro34]hPYY and hPYY(3-36) using the chloramine T method as described previously (Dumont et al., 1998), except that the column used for the high-performance liquid chromatography purification of iodinated peptides was the C18 Guard-Pak. The specific activity of radioligands was assumed to be of the theoretical value (2000 Ci/mmol).
Membrane Preparations. Membranes were prepared as described previously (Dumont et al., 1998). In brief, rats were killed by decapitation and their brains were rapidly removed and homogenized in a Krebs-Ringer phosphate (KRP) buffer, pH 7.4, composed of 120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM dextrose, and 25 mM NaHCO3 using a Brinkmann Polytron (at setting 6 for 15–20 s). Homogenates were centrifuged at 49,000 x g for 20 min; supernatants were discarded; and pellets were washed, resuspended, and recentrifuged twice.
Receptor Binding Assays. All binding assays were initiated by adding 100 µl of membrane preparations in a final volume of 500 µl of KRP containing 0.1% (w/v) BSA, 0.05% (w/v) bacitracin, radioligand, and unlabeled peptide or competitor as needed. Competition binding experiments were performed in the presence of 50 pM 125I-[hPP or 125I-Leu31,Pro34]hPYY and various competitors ([Leu31, Pro34]pNPY, [Leu31,Pro34]hPYY, rPP, hPP, [Ala31,Aib32]pNPY, [hPP(1-17), Ala31, Aib32]hNPY, [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP, GR231118, CP732925, CP760542, CP781214, JCF109, and CGP71683A) at concentrations ranging from 10–13 to 10–6 M.
Nonspecific binding was determined in the presence of 100 nM hPP or 1 µM [Leu31,Pro34]hPYY. After a 2-h incubation, the binding reaction was terminated by rapid filtration through Whatman Schleicher and Schuell #32 glass filters (previously soaked in 1.0% polyethylenimine) using a cell harvester filtering apparatus (Brandel Inc., Gaithersburg, MD). Filters were rinsed three times with 3 ml of ice-cold KRP, and the radioactivity remaining on filters was quantified using a gamma counter with 85% efficiency (Canberra Industries, Meriden, CT).
Quantitative Receptor Autoradiography. Receptor autoradiography was performed as described in detail previously (Dumont et al., 1998). In brief, rats were sacrificed by decapitation, and their brains were rapidly removed from the skull, frozen in 2-methylbutane at –40°C for 15 s, and then kept at –80°C until needed. Sections (20 µm) were obtained using a cryomicrotome at –17°C, mounted on gelatin-chrome-alum-coated slides, dried overnight in a desiccator at 4°C, and then kept at –80°C until use.
On the days of the experiments, adjacent coronal sections were preincubated for 60 min at room temperature in a KRP buffer at pH 7.4 and then incubated in a fresh preparation of KRP buffer containing 0.1% BSA, 0.05% bacitracin, 50 pM 125I-hPP, 125I-Leu31,Pro34]hPYY, or 125I-hPYY(3-36) in the presence and absence of 100 nM BIBO3304 (Y1 blocker), 1000 nM BIIE0246 (Y2 blocker), 10 nM GR231118 (Y4 blocker), and various Y5 receptor antagonists, such as JCF109, CGP71683A, CP732925, CP760542, and CP781214 (1000 nM). After a 2-h incubation, sections were washed four times, 1 min each in ice-cold KRP buffer, and then dipped in deionized water to remove salts and rapidly dried. Nonspecific binding was determined using 100 nM hPP, 1 µM [Leu31,Pro34]hPYY, or 1 µM hPYY(3-36). Incubated sections were apposed against Kodak BiomaxMR films (Eastman Kodak, Rochester, NY) for 7 to 14 days alongside radioactive standards. Films were developed and quantified as described in detail previously (Dumont et al., 1998).
All binding experiments were repeated three to six times (each in triplicate), and results (mean ± S.E.M.) are expressed as percentage of specific binding. Ki values (concentration of unlabeled competitor required to compete for 50% of specific bound radioligand) for the various competitors were calculated using GraphPad Prism (GraphPad Software Inc., San Diego, CA) for a one- or two-site model.
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Results |
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, 2004a) recognized a heterogeneous population of receptor binding sites in the mammalian brains. All three radioligands bind to the Y5 receptor subtype, although with different affinities (Michel et al., 1998; Dumont et al., 2004a). We investigated in detail the ligand selectivity profile of specific 125I-[Leu31,Pro34]hPYY binding in the presence of Y1 blockers using Y4 agonists, such as rPP and GR231118 (Lundell et al., 1995; Schober et al., 1998); selective Y5 agonists, including [Ala31,Aib32]pNPY, [hPP(1-17),Ala31,Aib32]hNPY, and [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP (Cabrele et al., 2001); and nonpeptide Y5 antagonists, such as CGP71683A (Criscione et al., 1998), JCF109 (Feletou et al., 1999), CP732925 (Elliott et al., 2003), and CP760542 and CP781214 (Yannielli et al., 2004). As shown in Fig. 1, Y5 agonists were able to compete with high affinity for specific 125I-[Leu31,Pro34]hPYY/BIBO3304-insensitive sites in rat brain membrane homogenates. Y5 antagonists also were potent competitors against specific 125I-[Leu31,Pro34]hPYY/ BIBO3304-insensitive sites (Fig. 1). However, Y5 receptor antagonists inhibited only up to 80 to 85% of specific 125I-[Leu31,Pro34]hPYY/BIBO3304-insensitive binding sites.
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Competition binding curves were analyzed in detail, and data were fitted to one- or two-site models. As shown in Table 1, competition curves against specific 125I-[Leu31, Pro34]hPYY/Y1-insensitive sites for [Leu31,Pro34]hPYY, hPP, GR231118, [Ala31,Aib32]pNPY, [hPP(1-17),Ala31,Aib32]hNPY, and [cPP(1-17), NPY(19-23),Ala31,Aib32,Gln34]hPP were best fitted to a two-site model (p < 0.05). In contrast, profiles obtained with Y5 antagonists, including GR231118, JCF109, CP732925, CP760524, and CP781214, were best fitted to a one-site model. In addition, competition curve analysis for Y5 receptor antagonists revealed that 11 to 19% of specific 125I-[Leu31,Pro34]hPYY/Y1-insensitive binding was also insensitive to Y5 antagonists (Table 1). Competition curves against specific 125I-hPP binding obtained with agonists also were best fitted to a two-site model (p < 0.05), except for pNPY, [Leu31,Pro34]pNPY, and hPYY(3-36) (Table 2). Curve-fitting analysis revealed that rPP and GR231118 recognized at least two populations of sites, high-(KH of 0.3 to 0.6 nM) and low (KL of 115 nM)-affinity sites (Table 2) with 30% of specific 125I-hPP binding belonging to KH. It is noteworthy that Y5 receptor antagonists competed against specific 125I-hPP binding with a best fit to a one-site model (Table 2). However, 20 to 29% of specific 125I-hPP binding was insensitive to Y5 antagonists. In addition, Y5 agonists such as [Ala31,Aib32]pNPY, [hPP(1-17), Ala31,Aib32]hNPY, and [cPP(1-17), NPY(19-23),Ala31,Aib32,Gln34]hPP competed against 125I-hPP binding with a best fit to a two-site model, with approximately 15% of specific 125I-hPP binding being insensitive these Y5 agonists (Table 2). Together, these data demonstrate that 125I-[Leu31,Pro34]hPYY in the presence of Y1 blockers and 125I-hPP recognized at least two populations of sites in rat brain membrane homogenates. One site is sensitive to Y5 receptor antagonists, whereas the other is not.
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We investigated next the discrete distribution of specific 125I-[Leu31,Pro34]hPYY binding sites insensitive to Y1 and Y5 blockers in the rat brain. Adjacent coronal rat brain sections were processed with 50 pM 125I-[Leu31,Pro34]hPYY in the presence of either 100 nM BIBO3304 to mask Y1 receptors; 100 nM BIBO3304 and 1000 nM CP732925 to mask both Y1 and Y5 sites; or 1000 nM [Leu31,Pro34]hPYY to determine nonspecific binding. As shown in Fig. 3, significant amounts of specific 125I-[Leu31,Pro34]hPYY binding resistant to Y1 and Y5 blockers were observed in the lateral septum, area postrema, and nucleus tractus solitarius. Quantitative receptor autoradiography revealed that significant amounts of specific 125I-[Leu31,Pro34]hPYY binding insensitive to Y1 and Y5 blockers are present in the lateral septum, various hypothalamic nuclei, hippocampus, nucleus tractus solitarius, area postrema, and dorsal motor nucleus of the vagus, whereas other structures contained only low levels of specific 125I-[Leu31,Pro34]hPYY/Y1- and Y5-insensitive sites (Fig. 4). Similar levels of inhibition was observed in these brain structures using any of the Y5 competitors studied here, namely, CGP71683A, JCF109, CP732925, CP760542, CP781214, and [hPP(1-17),Ala31,Aib32]hNPY (Fig. 4).
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). However and rather surprisingly, high levels of specific 125I-[Leu31,Pro34]hPYY/Y1- and Y5-insensitive sites were observed in the lateral septum (Fig. 3), whereas only very low levels of specific 125I-hPP/Y5-insensitive sites were found in this structure (Fig. 5). In contrast, although very low amounts of specific 125I-[Leu31,Pro34]hPYY/Y1 and Y5-insensitive sites were detected in the interpeduncular nuclei (Fig. 3), a much stronger signal was visualized using 125I-hPP in the presence of a Y5 blocker (Fig. 5). In few other brain structures such as the nucleus tractus solitarius and area postrema, similar levels of specific 125I-[Leu31,Pro34]hPYY/Y1- and Y5-insensitive sites and specific 125I-hPP/Y5-insensitive sites were detected (Figs. 3 and 5). To investigate this issue further, we evaluated specific 125I-hPP binding in the presence of 10 nM GR231118, a concentration known to fully mask putative Y4 receptor binding sites (Fig. 2) as well as residual specific 125I-hPP binding in the presence of 10 nM GR231118 and 1000 nM CP732925 (to mask both Y4 and Y5 sites). As shown in Fig. 6, significant amounts of specific 125I-hPP binding were visualized in the interpeduncular nuclei, nucleus tractus solitarius, and area postrema, even in the presence of 10 nM GR231118 and 1000 nM CP732925. These unexpected data prompted us to investigate in detail residual specific 125I-hPP binding in the presence of 10 nM GR231118 and different Y5 blockers, including CGP71683A, JCF109, CP732925, CP760542, CP781214, [hPP(1-17), Ala31,Aib32]hNPY, and PYY. Quantitative receptor autoradiography revealed that all Y5 molecules (agonists and antagonists) studied here were able to compete for similar amounts of specific sites labeled by 125I-hPP (Fig. 7). Most interestingly, high amounts of specific 125I-hPP binding insensitive to both GR231118 and various Y5 analogs were detected in the interpeduncular nucleus, nucleus tractus solitarius, area postrema and dorsal motor nucleus of the vagus, whereas moderate amounts were observed in the medial preoptic area, paraventricular nucleus of the hypothalamus, and vestibular nucleus (Fig. 7). All other regions contained low to very low levels of those sites (Fig. 7). Moreover, 1000 nM hPYY was not able to fully inhibit specific 125I-hPP labeling in the presence of GR231118 (Fig. 7), whereas residual specific 125I-[Leu31,Pro34]hPYY binding in the presence of Y1 and Y5 blockers was fully competed by 1000 nM PYY (Fig. 4).
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We have previously reported that 125I-hPYY(3-36) recognized at least two populations of sites, a Y2-sensitive site and Y2-insensitive site, and have postulated that 125I-hPYY(3-36)/Y2-insensitive sites represented the Y5 receptor subtype (Dumont et al., 2000b). The results obtained with 125I-hPP in the presence of Y4 and Y5 blockers prompted us to reinvestigate the specific labeling of 125I-hPYY(3-36) at the level of the area postrema, an area known to contain significant amounts of specific 125I-hPYY(3-36)/Y2-insensitive binding sites (Dumont et al., 2000b). As shown in Fig. 8, significant amounts of specific 125I-hPYY(3-36) binding is still visualized under Y2 and Y5 masking conditions in the area postrema, nucleus tractus solitarius, and dorsal motor nucleus of the vagus, in accordance with data reported above using 125I-hPP under Y4 and Y5 blocking conditions.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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NPY receptors have been characterized pharmacologically using various agonists and antagonists. The Y1 subtype is activated by NPY, PYY, [Leu31,Pro34]NPY, and [Leu31,Pro34]PYY, whereas C-terminal fragments and PPs are much less potent (Michel et al., 1998; Dumont et al., 2004a). Selective Y1 receptor antagonists have been developed, including BIBP3226 (Rudolf et al., 1994), BIBO3304 (Wieland et al., 1998), and GR231118 (Daniels et al., 1995). The Y2 subtype has high affinity for NPY, PYY and their C-terminal fragments [PYY(3-36) and NPY(13-36)], whereas [Leu31,Pro34]NPY, [Leu31,Pro34]PYY, and PPs are much less potent (Michel et al., 1998; Dumont et al., 2004a). Two nonpeptide Y2 receptor antagonists have been characterized thus far, BIIE0246 (Doods et al., 1999) and JNJ-5207787 (Bonaventure et al., 2004). The Y4 subtype is characterized by its high affinity for PPs and GR231118 (as an agonist; Michel et al., 1998). The Leu31Pro34-substituted analogs of NPY and PYY are also potent agonists on the Y4 subtype (Gehlert et al., 1996). This receptor subtype can be activated by PYY and NPY, but with much lower potencies than the PPs (Lundell et al., 1995). As to the Y5 subtype, NPY, PYY, [Leu31,Pro34]NPY, [Leu31,Pro34]PYY, NPY(2-36), PYY(3-36), and hPP display potent agonist activities, whereas NPY(13-36), PYY(13-36), rPP, and GR231118 are much less active (Michel et al., 1998; Dumont et al., 2004a). Selective Y5 receptor agonists have been developed, including [Ala31,Aib32]NPY, [hPP(1-17),Ala31,Aib32]hNPY, and [cPP(1-7), NPY(19-23),Ala31,Aib32,Gln34]hPP (Cabrele et al., 2001). In addition, various nonpeptide Y5 antagonists have been characterized including CGP71683A (Criscione et al., 1998), JCF109 (Feletou et al., 1999), CP732925 (Elliott et al., 2003), and CP760542 and CP781214 (Yannielli et al., 2004). Hence, a variety of selective peptide and nonpeptide molecules are now available to selectively study each of the cloned receptor subtype.
We previously suggested that specific 125I-[Leu31,Pro34]hPYY/Y1-insensitive sites represented the Y5 receptor subtype, based on competition profiles of various agonists and antagonists (Dumont et al., 1998) and the finding that BIBO3304 (Y1 antagonist) and CGP71683A (Y5 antagonist) were able to compete for 75 and 25% of specific 125I-[Leu31,Pro34]hPYY binding, respectively (Dumont et al., 2000a). However, it was not possible to exclude a possible contribution of the Y4 subtype because 125I-[Leu31, Pro34]PYY has rather high affinity for this subtype (Gehlert et al., 1997). In the present study, we demonstrated that 125I-[Leu31,Pro34]hPYY recognized at least three distinct classes of sites. As expected, the first two represent the Y1 and Y5 subtypes. The third class is unlikely to be only the Y4 subtype on the basis of its distribution profile in the rat brain.
The discrete localization of the Y4 subtype has previously been investigated using different radioligands including 125I-hPP and 125I-rPP (Trinh et al., 1996), 125I-bovine PP (Gehlert et al., 1997) and 125I-GR231118 (Dumont and Quirion, 2000; Schober et al., 2000). Using these various probes, it was shown that specific 125I-PP binding sites (the Y4 subtype) are particularly enriched in the interpeduncular nuclei, nucleus tractus solitarius, and area postrema of the rat brain. In the present study, specific 125I-[Leu31,Pro34]PYY binding insensitive to Y1 and Y5 antagonists (hypothesized to be Y4-like) demonstrated much stronger binding levels than specific 125I-hPP/Y5-insensitive (also hypothesized to be Y4-like) sites in various regions such as the lateral septum. More surprisingly, clearly distinct localization patterns were seen in some areas, including the interpeduncular nucleus, which is only enriched in specific 125I-hPP/Y5-resistant binding sites. Together, these data suggest the existence of additional NPY/PP receptor subtype(s) in the rat brain.
This hypothesis is supported further by comparative anatomical data obtained using 125I-hPP and 125I-hPYY(3-36). Both radioligands revealed the presence in the area postrema of sites that are insensitive to Y4 and Y5 blockers in the case of 125I-hPP and Y2 and Y5 antagonists for 125I-hPYY(3-36). Significant amounts of specific 125I-hPP sites resistant to Y4 and Y5 blockers were also detected in the medial preoptic area, paraventricular nucleus of the hypothalamus, interpeduncular nuclei, nucleus tractus solitarius, and dorsal motor nucleus of the vagus. It is noteworthy that, using triple Y1, Y2, and Y4 knockout mice, Lin et al. (2005) showed that residual specific 125I-PYY binding could be detected in the hippocampus even after blocking the Y5 subtype and in the absence of y6 receptor mRNA.
Can the atypical binding sites characterized and visualized in the present study represent receptor dimers or the elusive Y3 subtype? It was recently shown that Y1, Y2, Y4, and Y5 receptors can form homodimers (Berglund et al., 2003b; Dinger et al., 2003). The possible existence of Y1 and Y5 heterodimers has also been suggested (Schober et al., 2003). However, there is no evidence that once NPY receptors form homo- or heterodimers, they display distinct pharmacological profiles compared with the native monomeric receptor (Schober et al., 2003). Regarding the Y3 subtype, the existence of this receptor was proposed on the basis of cardiovascular and cAMP responses induced only by NPY and its homologues; PYY and PP derivatives being inactive (Ny and Grundemar, 1997; Glaum et al., 1997). Specific sites characterized in the present study clearly displayed high affinities for both PYY and PPs, excluding the Y3 subtype as the one characterized here. It is noteworthy that elaborated phylogenic studies suggested the existence of additional NPY receptor subtypes, in fact up to eight to nine subtypes in total (Larhammar, 1996; Larhammar and Salaneck, 2004).
At the functional level, the high expression of atypical specific [125]PYY(3-36) and 125I-hPP binding sites in the area postrema is of interest because this region is devoid of a mature blood-brain barrier. Blood-borne PYY(3-36) (Batterham et al., 2002) and PPs (Batterham et al., 2003) were shown to be potent inhibitors of food intake. It was also suggested that the peripheral effects of PYY(3-36) on food intake were mediated by the Y2 receptor subtype because peripheral administration of PYY(3-36) had no effect on this behavior in Y2 knockout animals (Batterham et al., 2002). However, this hypothesis cannot explain the effect obtained with i.v. injections of PPs because these peptides have no significant affinity for the Y2 receptor subtype. The novel class of binding sites reported here represents an interesting alternative hypothesis, especially because the area postrema, nucleus tractus solitarius, and dorsal motor nucleus of the vagus are known to be involved in the regulation of food intake and cardiovascular parameters (Dumont et al., 2000c)
In summary, competition binding experiments in rat brain membrane homogenates demonstrated that specific 125I-[Leu31,Pro34]PYY/Y1-insensitive and specific 125I-hPP binding sites are competed by both Y4 and Y5 ligands with competition binding profiles best fitted to a two-site model. Receptor autoradiography revealed the differential localization of specific 125I-[Leu31,Pro34]PYY and 125I-hPP sites in the presence of Y1, Y4, and Y5 blockers in the rat brain. Enrichments in specific 125I-[Leu31,Pro34]PYY/Y1 and Y5-resistant sites are observed in the lateral septum, nucleus tractus solitarius, and area postrema, whereas high levels of specific 125I-hPP/Y4 and Y5-insensitive sites are found in the interpeduncular nucleus, nucleus tractus solitarius, area postrema, and dorsal motor nucleus of the vagus. Residual specific 125I-PYY(3-36)/Y2 and Y5-insensitive sites were also detected in the area postrema, among other regions. Together, these data suggest the existence of additional subtype of NPY binding sites in the rat CNS, beside the well established Y1, Y2, Y4, and Y5 receptors.
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Footnotes |
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ABBREVIATIONS: NPY, neuropeptide Y; PYY, peptide YY; PP, pancreatic polypeptide; hPYY, human peptide YY; hPP, human pancreatic polypeptide; cPP, chicken pancreatic polypeptide; BIBP3226, R-N2-(diphenylacetyl)-N-(4-hydroxyphenyl)-methyl argininamide; BIBO3304, (R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphenylacetyl)-argininamide trifluoroacetate; CNS, central nervous system; CGP71683A, N1-[(4-([(4-amino-2-quinazolinyl)amino]methyl)cyclohexyl)methyl]-1-naphtale-nesulfonamide; rPP, rat pancreatic polypeptide; GR231118, homodimeric Ile-Glu-Pro-Dpr-Tyr-Arg-Leu-Arg-Tyr-CONH2; pNPY, porcine neuropeptide Y; BIIE0246, (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]-ethyl]-argininamid; CP732926, 3-[2-[6-(2-tert-butoxyethoxy)pyridin-3-yl]-1H-imidazol-4-yl]benzonitrile hydrochloride salt; JCF109, ([(naphtalen-2-ylmethyl)-amino]-methyl)-2-nitro-benze-nesulfonamide; BSA, bovine serum albumin; KRP, Krebs-Ringer phosphate; CGP71683A, trans-naphtalene-1-sulfonic acid [4-[(4-amino-quinazolin-2-ylamino)-methyl]-cyclohexylmethyl]-amide hydrochloride.
Address correspondence to: Dr. Rémi Quirion, Douglas Hospital Research Center, 6875 Blvd. LaSalle, Montréal (Verdun), QC, Canada H4H 1R3. E-mail: remi.quirion{at}douglas.mcgill.ca
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References |
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Batterham RL, Le Roux CW, Cohen MA, Park AJ, Ellis SM, Patterson M, Frost GS, Ghatei MA, and Bloom SR (2003) Pancreatic polypeptide reduces appetite and food intake in humans. J Clin Endocrinol Metab 88: 3989–3992.
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