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Keywords:

  • Receptor subtype;
  • NPY;
  • binding assay;
  • bioassay;
  • antagonist

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. List of Abbreviations used in figures
  8. Acknowledgments
  9. References
  • BIIE0246, a newly synthesized non-peptide neuropeptide Y (NPY) Y2 receptor antagonist, was able to compete with high affinity (8 to 15 nM) for specific [125I]PYY3–36 binding sites in HEK293 cells transfected with the rat Y2 receptor cDNA, and in rat brain and human frontal cortex membrane homogenates.

  • Interestingly, in rat brain homogenates while NPY, C2-NPY and PYY3–36 inhibited all specific [125I]PYY3–36 labelling, BIIE0246 failed to compete for all specific binding suggesting that [125I]PYY3–36 recognized, in addition to the Y2 subtype, another population of specific NPY binding sites, most likely the Y5 receptor.

  • Quantitative receptor autoradiographic data confirmed the presence of [125I]PYY3–36/BIIE0246-sensitive (Y2) and-insensitive (Y5) binding sites in the rat brain as well as in the marmoset monkey and human hippocampal formation.

  • In the rat vas deferens and dog saphenous vein (two prototypical Y2 bioassays), BIIE0246 induced parallel shifts to the right of NPY concentration-response curves with pA2 values of 8.1 and 8.6, respectively. In the rat colon (a Y2/Y4 bioassay), BIIE0246 (1 μM) completely blocked the contraction induced by PYY3–36, but not that of [Leu31,Pro34]NPY (a Y1, Y4 and Y5 agonist) and hPP (a Y4 and Y5 agonist). Additionally, BIIE0246 failed to alter the contractile effects of NPY in prototypical Y1in vitro bioassays.

  • Taken together, these results demonstrate that BIIE0246 is a highly potent, high affinity antagonist selective for the Y2 receptor subtype. It should prove most useful to establish further the functional role of the Y2 receptor in the organism.

British Journal of Pharmacology (2000) 129, 1075–1088; doi:10.1038/sj.bjp.0703162


Abbreviations:
BIBO3304

((R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphenylacetyl)-argininamide trifluoroacetate)

BIBP3226

R-N2-(diphenylacetyl)-N-(4-hydroxyphenyl)-methyl argininamide

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

SR120819A

(R,R)-(1-[2-[2-(2-naphthylsulphamoyl)-3-phenylpropionamido]-3-[4-[N-[4-(dimethylaminomethyl)-cis-cyclohexylmethyl] amidino] phenyl] propionyl]-pyrrolidine)

LY357897

1-(1-[3-((3s)(3-piperidyl))-propyl]-2-[(4-chlorophenoxyl)-methyl]indol-3-yl]-2-(4-piperidylpiperidyl)ethan-1-one

GR231118

homodimeric Ile-Glu-Pro-Dpr-Tyr-Arg-Leu-Arg-Tyr-CONH2

h

human

HEK293

human embryonic kidney cells

KRP

Krebs Ringer phosphate buffer

NPY

neuropeptide Y, p, porcine

PP

pancreatic polypeptide

PYY

peptide YY

r

rat

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. List of Abbreviations used in figures
  8. Acknowledgments
  9. References

Several studies have addressed the physiological functions of neuropeptide Y (NPY) and its homologues, peptide YY (PYY) and the pancreatic polypeptides (PP) in the central nervous system (CNS) (for reviews see: Colmers & Bleakman, 1994; Dumont et al., 1992; Gehlert, 1998; Heilig & Widerlov, 1995; Inui, 1999; Kalra & Crowley, 1992; Wahlestedt & Reis, 1993; Vezzani et al., 1999) and demonstrated a broad range of effects. For example, these peptides stimulate feeding behaviours and water consumption (Jolicoeur et al., 1991a,1991b; Morley & Flood, 1989; Stanley & Leibowitz, 1984), facilitate learning and memory processes (Flood et al., 1987), inhibit glutamatergic excitatory synaptic transmission at Schaffer collaterals and mossy fibres (Colmers & Bleakman, 1994; Klapstein & Colmers, 1993), have anticonvulsive properties (Klapstein & Colmers, 1997; Woldbye, 1998; Vezzani et al., 1999), modulate neuroendocrine secretions (Kalra & Crowley, 1984; 1992) and are anxiolytics (Heilig et al., 1993). Several of these effects appear to be physiologically relevant based on data obtained using NPY antibody, antisense oligonucleotides or knockout mice (for more details see Dumont et al., 2000a).

Thus far, five classes of receptors have been cloned and classified as the Y1, Y2, Y4, Y5 and y6 subtypes on the basis of their molecular and pharmacological profiles (Michel et al., 1998). All NPY receptor subtypes are expressed in several species including man (Larhammar, 1996), except for the y6 receptor subtype which is not expressed in the rat (Burkhoff et al., 1998) while in human and primates, the translated protein is a non-functional receptor due to a truncation from the half of the sixth transmembrane domain (Gregor et al., 1996; Matsumoto et al., 1996). The respective structure-activity relationships of each of these receptors has been established using several analogues and fragments of NPY, PYY and PPs (Blomqvist & Herzog, 1997; Michel et al., 1998). However, most of the currently available agonists such as [Pro34]NPY, [Leu31,Pro34]NPY, [Leu31,Pro34]PYY and PYY3–36, first reported as selective for the Y1 or Y2 receptors (Dumont et al., 1994; Fuhlendorff et al., 1990; Schwartz et al., 1990) are now known to also have significant affinities for the more recently cloned Y4 and Y5 subtypes (Blomqvist & Herzog, 1997; Dumont et al., 1998a; Gehlert et al., 1996a,1996b; Gerald et al., 1996; Michel et al., 1998). Accordingly, a series of agonists must be used to tentatively establish the possible role of a given receptor subtype in mediating an effect induced by NPY and its homologues (Michel et al., 1998).

The use of highly selective antagonists is often preferable to precisely classify receptor subtypes. Over the past few years, few peptides and especially non-peptide NPY antagonists have been developed. The first non-peptide Y1 antagonist to be reported was R-N2-(Diphenylacetyl)-N-(4-hydroxyphenyl)-methyl argininamide, known as BIBP3226 (Rudolf et al., 1994). This antagonist has been extensively studied and most data have shown that BIBP3226 behaves as a competitive, selective and specific Y1 receptor antagonist in various binding assays as well as in in vitro and in vivo bioassays (Abounader et al., 1995; Bergdahl et al., 1996; Doods et al., 1995; 1996; Jacques et al., 1995; Lundberg & Morin, 1995; Lundberg et al., 1996; Malmstrom et al., 1997; Nilsson et al., 1996a,1996b; Racchi et al., 1996; Rudolf et al., 1994; Tough & Cox, 1996; Wieland et al., 1995; Zukowska-Grojec et al., 1996), without any significant affinity for the Y2 (Doods et al., 1996; Dumont et al., 1998a; Gehlert et al., 1996c; Gerald et al., 1996; Jacques et al., 1995; Rudolf et al., 1994), Y4 (Doods et al., 1996; Gehlert et al., 1996a; Gerald et al., 1996) and Y5 (Doods et al., 1996; Dumont et al., 1998a; Gerald et al., 1996) receptors. More recently, ((R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphenylacetyl)-argininamide trifluoroacetate) or BIBO3304 was reported to be highly selective for the human and rat Y1 receptors having a 10 fold greater affinity for this subtype than BIBP3226 (Dumont et al., 2000b; Wieland et al., 1998). Other Y1 antagonists including (R,R) -(1-[2-[2-(2-naphthylsulphamoyl) -3- phenylpropionamido]-3-[4-[N-[4-(dimethylaminomethyl)-cis-cyclohexylmethyl] amidino] phenyl] propionyl]-pyrrolidine) known as SR120819A (Serradeil-Le Gal et al., 1995) and 1-(1-[3-((3s)(3-piperidyl))-propyl]-2-[(4-chlorophenoxyl) -methyl] indol-3-yl] -2-(4-piperidylpiperidyl)ethan-1-one known as LY357897 (Hipskind et al., 1997) have also been reported although not as extensively studied as BIBP3226 and BIBO3304. Additionally, a Y1 peptide antagonist, homodimeric Ile-Glu-Pro-Dpr-Tyr-Arg-Leu-Arg-Tyr-CONH2, firstly known as 1229U91 (Daniels et al., 1995a) or GW1229 (Bitran et al., 1997) and now as GR231118 (Michel et al., 1998; Parker et al., 1998) has been rather extensively investigated. GR231118 was first characterized as a specific Y1 receptor antagonist on the basic of its selective blockade of Y1-like vs Y2-like effects (Bitran et al., 1997; Daniels et al., 1995a,1995b; Leban et al., 1995). However, more recent data have shown that GR231118 is also a potent agonist on the Y4 (Dumont & Quirion, 2000; Kanatani et al., 1998; Parker et al., 1998; Schober et al., 1998) and y6 (Parker et al., 1998) receptors with some weak agonistic properties on the Y5 receptor (Dumont et al., 1998a; Kanatani et al., 1998; Parker et al., 1998). Recently, few non-peptide Y5 antagonists have been characterized including [(4-{[(4-amino-quinazolin-2-yl)amino]methyl}-cyclohexyl)methyl](naphthyl-suphonyl)amine, known as CGP71683A (Criscione et al., 1998a; Dumont et al., 2000b) and L-152804 (Kanatani et al., 1997). Preliminary studies seem to demonstrate their potent antagonistic properties and selectivity for the Y5 receptor (Criscione et al., 1998; Kanatani et al., 1997) although further characterization is certainly warranted.

Considering the high amounts of specific [125I]PYY3–36/Y2-like binding sites detected in the brain of various species, especially in the hippocampal formation (Dumont et al., 1996;1998b) and the possible implication of the Y2 receptor subtype in various biological effects induced by NPY (for review see Colmers & Bleakman, 1994; Dumont et al., 2000a; Gehlert, 1998; Vezzani et al., 1999), it was deemed critical to develop selective Y2 receptor antagonists. One group has proposed T4-[NPY33–36]4 as potent Y2 antagonist (Grouzmann et al., 1997). However, subsequent studies by this group (Grouzmann et al., 1998) and ours (Pheng et al., 1999) demonstrated its rather low affinity. Most recently, Doods et al. (1999; 5th International NPY Meeting, Cayman Island, April 17–22, 1999) reported on the development of 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) as the first potent non-peptide Y2 antagonist devoid of apparent agonistic or antagonistic activities for the Y1, Y4 and Y5 receptors.

In the present study, we investigated in details the profile of BIIE0246 for various NPY receptors using cloned transfected receptors in HEK 293 cells, rat and human brain membrane homogenates, quantitative receptor autoradiography in mammalian CNS as well as a variety of in vitro bioassays. Our data clearly demonstrate that BIIE0246 is the first potent and highly selective Y2 receptor antagonist to be developed.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. List of Abbreviations used in figures
  8. Acknowledgments
  9. References

Materials

Male Sprague Dawley CD rats (200–250 g) and Albino New-Zealand rabbits of either sex (1.5–2.0 Kg) were obtained from Charles River Canada (St-Constant, Québec, Canada). Mongrel dogs of either sex (20–50 Kg) were obtained from the Laboratory of the Animal Protection Branch (Sherbrooke, QC, Canada). All animals were kept on a 12 h light-dark cycle (light on at 07:00) in temperature and humidity controlled rooms. Animals were fed with standard laboratory chow and had access to tap water ad libitum. Animal care was according to protocols and guidelines approved by McGill University, University of Sherbrooke and the Canadian Council of Animal Care. Human cerebral arteries reactivity studies were conducted in small ramification of middle cerebral arteries obtained post-morterm from patients with no cerebrovascular pathologies following approval of the research ethic committees from the Douglas Hospital or the Montreal Neurological Institute. Sanofi Recherche (Montpellier, France) generously provided marmoset (C. jacchus) monkey brains. Human frontal cortex and brain blocks were provided by the Douglas Hospital Research Centre Brain Bank from tissues obtained according to protocols and methodology described elsewhere (Quirion et al., 1987). These brains were obtained from human with post-morterm delays varying between 8–23 h in which the neuropathological examinations revealed no evidence of neurological disorders, significant cell losses, plaques, neurofibrillary tangles or excessive gliosis.

Analogues and fragments of human (h) PYY, porcine (p) NPY and hPP were synthesized as previously described (Forest et al., 1990). BIBP3226, BIBO3304 and BIIE0246 were generously provided by Boerhinger Ingelheim (Germany) while GR231118 (first known as 1229U91 or GW1229) and CGP71683A were a gift from Glaxo Wellcome (Research Triangle Park NC, U.S.A.) and Servier (Paris, France), respectively. T4-[NPY33–36]4 was purchased from Dr E. Grouzmann (Lausanne, Switzerland) and C2-NPY was obtained from Dr M. Aubert (Geneva, Switzerland). Bovine serum albumin (BSA) and Iodine-125 were obtained from ICN Pharm. Canada Ltd. (Montréal, Québec, Canada) and bacitracin was purchased from Sigma Chemical (St-Louis, MI, U.S.A.). Schleicher and Schuell #32 glass filters were obtained from Xymotech (Montréal, Québec, Canada). [3H]Hyperfilms and 125I-microscale standards were purchased from Amersham (Mississauga, Ontario, Canada). All tissue culture media, antibiotics and reagents were obtained from Gibco-BRL (Burnington, Ontario, Canada). The rat Y1, Y2, Y4 and Y5 receptor cDNA were generously provided by Dr H. Herzog (Sydney, Australia). The expression vector, pcDNA3, was purchased from Invitrogen (San Diego, CA, U.S.A.). All other chemicals were of analytical grade and obtained from Fisher Scientific (Montreal, QC, Canada) or Sigma Chemical (St-Louis, MI, U.S.A.).

Iodine-125 was incorporated into the tyrosine residue of [Leu31,Pro34]PYY, PYY3–36, hPP and GR231118 using the chloramine T method as previously described (Dumont et al., 1995; Dumont & Quirion, 2000) and the specific activity was assumed to be of the theoretical value (2000 Ci mmol−1).

Membrane preparations

Membranes were prepared as previously described (Dumont et al., 1995). Briefly, rats were killed by decapitation and their brains rapidly removed and homogenized in a Krebs Ringer phosphate (KRP) buffer at pH 7.4 of the following composition (mM: NaCl 120, KCl 4.7, CaCl2 2.2, KH2PO4 1.2, MgSO4 1.2, dextrose 5.5 and NaHCO3 25 using a Brinkman polytron (at setting 6 for 15–20 s). Human frontal cortex membrane homogenates were prepared as described elsewhere (Jacques et al., 1997). Homogenates were centrifuged at 49,000×g for 20 min, supernatants discarded and pellets washed, resuspended, and recentrifuged twice. Protein concentration was determined with BSA as the standard (Bradford, 1976).

Transfected cells

HEK 293 cells were maintained in Dulbecco's modified Eagle medium (D-MEM) supplemented with 10% foetal calf serum and antibiotics (penicillin G sodium, streptomycin sulphate and amphotericin B). Cultured cells were transfected with either of the rat Y1, Y2, Y4 or Y5 receptor cDNA using a calcium phosphate method (Tong et al., 1995). Briefly, 125 μl of 2.5 M calcium phosphate was added to 1.125 ml water containing 50 μg of either rat Y1, Y2, Y4 or Y5 receptor cDNA which was previously inserted in expressing pcDNA3 vectors and was slowly mixed with 1.25 ml 2× HEPES buffer at pH 7.05 and left at room temperature for 20 min. The mixture was added to a 150 mm dish containing HEK 293 cells at 30% confluent and returned to the incubator. The medium was changed the next morning. Forty-eight hours later, cells were washed with KRP buffer pH 7.4 and scraped. Detached cells were then centrifuged at 400×g for 10 min and the pellet washed with KRP buffer (pH 7.4), recentrifuged twice, and resuspended in 8 ml of KRP buffer pH 7.4 and used for receptor binding assay.

Binding assays

All binding assays were initiated by adding 100 μl of membrane or cell preparations in a final volume of 500 μl of KRP containing 0.1% (w v−1) BSA, 0.05% (w v−1) bacitracin radiolabelled probes and unlabelled peptide or competitor as needed. Isotherm saturations were performed in the presence of increasing concentrations of radiolabelled probes while competition binding experiments were performed using 30–35 pM of radiolabelled probes in the presence and absence of various competitors at concentrations ranging from 10−12–10−6 M. In the rat brain homogenates, Y1-like and Y2-like receptors were studied using [125I[Leu31,Pro34]PYY and [125I]PYY3–36, respectively and as previously described (Dumont et al., 1995). [125I]GR231118 and [125I][Leu31,Pro34]PYY were used in HEK 293 cells transfected with rat Y1 and Y5 receptor cDNA, respectively. Binding in HEK 293 cells transfected with rat Y2 and Y4 receptor cDNA was performed using [125I]PYY3–36 and [125I]hPP, respectively. Non-specific binding was determined in the presence of 1 μM pNPY. Following a 2 h incubation, the binding reaction was terminated by rapid filtration through Schleicher and Schuell #32 glass filters (previously soaked in 1.0% polyethyleneimine) using a cell harvester filtering apparatus (Brandel Instruments, Gaithersburg, MD, U.S.A.). Filters were rinsed three times with 3 ml of cold KRP and the radioactivity remaining on filters was quantified using a gamma counter with 85% efficiency (Packard Instruments).

All binding experiments were repeated three to six times, each in triplicate, and results expressed as percentage of specific binding (competition) or fmol (saturation). Kd values (i.e. the concentration of radioligand needed to occupy 50% of the total receptor population) were calculated from data obtained in saturation isotherm binding experiments using GraphPad Prism software (GraphPad Software Inc. San Diego, CA, U.S.A.) with a fit to a one site hyperbola curve. IC50 values (i.e. concentration of unlabelled peptide required to compete for 50% of specific binding of the radioligand) of the various peptides and antagonists were calculated from the competition binding assays data using the GraphPad Prism software with a fit to a sigmoidal dose response curve.

Quantitative receptor autoradiography

Receptor autoradiography was performed as described in detail elsewhere (Dumont et al., 1996; 1998a,1998b; Jacques et al., 1997). All 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 for 120 min in a fresh preparation of KRP buffer containing 0.1% BSA, 0.05% bacitracin, 30 pM [125I]PYY3–36 and various concentrations of BIIE0246 (10−10–10−5 M). Following a 2 h incubation, sections were washed four times, 2 min each in ice-cold KRP buffer then dipped in deionized water to remove salts and rapidly dried. Non-specific binding was determined using 1 μM NPY. Incubated sections were apposed against 3H-Hyperfilms for 6 days alongside radioactive standards. Films were developed and quantified as described in detail elsewhere (Dumont et al., 1996; 1998a).

In vitro bioassays

The rabbit (Cadieux et al., 1993) and dog (Pheng et al., 1997) saphenous veins, the rat vas deferens (Martel et al., 1990; Dumont et al., 1994), the rat colon (Pheng et al., 1999) and human cerebral arteries (Abounader et al., 1995) were prepared as described in details elsewhere. Concentration-response curves to NPY were generated by the cumulative addition of peptides for the rabbit saphenous vein and human cerebral arteries (NPY-induced contractions), and the rat vas deferens (NPY-inhibition of electrically stimulated twitch response) while a non-cumulative manner was used for the rat colon (NPY-induced contraction). In these tissues, the antagonistic properties of BIIE0246 were investigated by applying various concentrations or a single maximal concentration (1 μM) of BIIE0246 10 min prior to NPY. In the dog saphenous vein, the ability of BIIE0246 to block the contractile effects of NPY was investigated by the cumulative addition of BIIE0246 on tissues pre-contracted with NPY.

Concentration-response curves were constructed by plotting the molar concentration of NPY versus response expressed as percentage of the maximal response. From these plots, EC50 values were calculated by non-linear regression analysis (sigmoidal dose-response curve). EC50 values were calculated from each individual curve and the mean±s.e.mean was calculated from these data for the rat vas deferens, the rabbit saphenous vein and the human cerebral arteries. For each concentration of antagonist used, concentration-ratio was calculated by dividing the EC50 value for NPY in the presence of the antagonist by the EC50 obtained in the absence of the blocker. Schild plots were constructed and linear regression used to determine the x-intercept (pA2 value). For the dog saphenous vein, the pA2 value was determined as the concentration of BIIE0246 required to reduce by 50% the contractile effects of NPY.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. List of Abbreviations used in figures
  8. Acknowledgments
  9. References

Receptor binding assays in HEK 293 cells transfected with the rat Y2 receptor cDNA demonstrated that BIIE0246 competed with high affinity (IC50=15±3 nM) against specific [125I]PYY3–36 binding sites (Table 1). In contrast, BIIE0246, at concentrations up to 10 μM, failed to compete for significant amounts of specific [125I]GR231118, [125I]hPP and [125I][Leu31,Pro34]PYY binding sites in HEK 293 cells transfected with the rat Y1, Y4 or Y5 receptor cDNA, respectively (Table 1). On the other hand, the Y1 antagonists, BIBP3226 and BIBO3304 were potent competitors in HEK 293 cells expressing the Y1 but not Y2, Y4 and Y5 subtypes (Table 1) while the Y5 antagonist, CGP71683A, had high affinity in HEK 293 cells transfected with the rat Y5 receptor cDNA and GR231118 was a potent competitor for the Y1 and Y4 receptor binding sites expressed in HEK 293 cells (Table 1).

Table 1. Competition binding parameters of various antagonists of the NPY family against either [125I]GR231118, [125I]PYY3–36, [125I]hPP or [125I][Leu31,Pro34]PYY binding in HEK 293 cells transfected with the rat Y1, Y2, Y4 or Y5 receptor cDNAThumbnail image of

In rat brain homogenates, BIIE0246 was able to compete for specific [125I] PYY3–36 binding sites with an affinity similar to that observed in HEK 293 cells transfected with the rat Y2 receptor cDNA (Figure 1; Table 2). Specific [125I][Leu31,Pro34]PYY binding was not competed by BIIE0246 at concentrations up to 1 μM (Table 2). BIIE0246 was about 70 times more potent to inhibit [125I] PYY3–36 binding and possessed higher selectivity than T4-[NPY33–36]4, a purported Y2 peptide antagonist (Figure 1; Table 1). In contrast to pNPY, C2-NPY and hPYY3–36 which all competed for 100% of specifically bound [125I]PYY3–36 (as established using 1 μM pNPY), BIIE0246 was able to inhibit only up to 95% of the labelling (P<0.05; Figure 1), suggesting that [125I]PYY3–36 could recognize an additional population of binding sites in the rat brain.

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Figure 1. Competition binding profile of pNPY, C2-NPY, hPYY3–36, BIIE0246 and T4-[NPY33–36]4 against specific [125I]PYY3–36 binding sites in rat brain membrane preparations. Data represent the mean±s.e.mean of 4–6 determinations, each performed in triplicate.

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Table 2. Comparative binding parameters of porcine (p) NPY, human (h) PYY, their C-terminal fragments and BIBP3226 and BIBO3304 (two Y1 non-peptidergic antagonists), T4-[NPY33–36]4 (a purported Y2 peptide antagonist) and BIIE0246 (a Y2 non-peptidergic antagonist) against [125I][Leu31,Pro34]PYY and [125I]PYY3–36 binding sites in a rat brain and human frontal cortex membrane homogenatesThumbnail image of

The existence and the distribution of specific [125I]PYY3–36 binding sites that are highly sensitive or not to BIIE0246 was established next using quantitative receptor autoradiography. Adjacent coronal rat brain sections were incubated with [125I]PYY3–36 in the presence of increasing concentration (0.1 nM–10 μM) of BIIE0246 and demonstrated that specific [125I]PYY3–36 binding was fully competed by BIIE0246 in most brain structures (Figure 2). However, the quantitative analysis of [125I]PYY3–36 labelling revealed that BIIE0246 failed to compete for all specific [125I]PYY3–36 binding sites in few regions such as the external plexiform layer of the olfactory bulb, the anterior olfactory nucleus, the olfactory tubercle, the dorsal part of the lateral septum, the CA3 subfield of the ventral hippocampus and the area postrema (Figure 3). The specific [125I]PYY3–36 binding sites resistant to the Y2 receptor antagonist may represent the Y5 subtype. In HEK 293 cells expressing the rat Y5 receptor gene, [125I][Leu31,Pro34]PYY, [125I]PYY3–36 and [125I]hPP were all able to recognize the expressed Y5 receptor protein (Table 3). However, [125I][Leu31,Pro34]PYY has higher affinity than [125I]PYY3–36 and [125I]hPP for the transfected Y5 receptor subtype (Table 3). In fact, at concentrations used in receptor binding and autoradiographic studies, [125I][Leu31,Pro34]PYY specifically labelled three to four times more Y5 sites than [125I]PYY3–36 (data not shown). This may explain the lower signal detected using [125I]PYY3–36/BIIE0246-insensitive sites (Figure 2) as compared to Y5 receptors characterized as specific [125I][Leu31,Pro34]PYY/Y1 antagonists-insensitive sites (Dumont et al., 1998a; 2000b).

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Figure 2. Photomicrographs of the autoradiographic distribution of [125I]PYY3–36 binding sites in presence and absence of various concentrations of BIIE0246 in the rat brain. Adjacent coronal rat brain sections were incubated in the presence of 30 pM [125I]PYY3–36 and in the presence of BIIE0246 from 1–1000 nM. Non-specific binding was determined in the presence of 1 μM pNPY. See list of abbreviations for anatomical identification. Scale bar represents 10 mm.

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Figure 3. Quantitative autoradiographic data of [125I]PYY3–36 in the presence of increasing concentrations of BIIE0246 (0.1–10,000 nM) in various rat brain regions. See list of abbreviations for anatomical identification.

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Table 3. Comparative affinities (Kd) of various radioligands for the Y1, Y2, Y4 and Y5 receptors expressed in HEK 239 cells transfected with their respective receptor cDNAThumbnail image of

Further characterization of specific [125I]PYY3–36 binding sites sensitive to BIIE0246 was done in other species. In human frontal cortex homogenates, BIIE0246 was able to compete against specific [125]PYY3–36 binding with an apparent affinity of 8 nM (Table 1). Additionally, autoradiographic studies demonstrated that specific [125I]PYY3–36 binding was fully inhibited by BIIE0246 (1 μM) in most areas of the marmoset monkey (Figure 4) and human (Figure 5) brains. However, some regions such as the hippocampal formation revealed the existence of [125I]PYY3–36/BIIE0246-insensitive sites in both species (Figures 4 and 5; Table 4).

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Figure 4. Photomicrographs of the autoradiographic distribution of [125I]PYY3–36 binding sites in the marmoset monkey brain. Adjacent coronal brain sections were incubated with 30 pM [125I]PYY3–36 and in the presence of 1000 nM BIIE0246. Non-specific binding was determined in the presence of 1 μM pNPY. See list of abbreviations for anatomical identification. Scale bar represents 10 mm.

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Figure 5. Photomicrographs of the autoradiographic distribution of [125I]PYY3–36 binding sites in the human brain hippocampal area. Adjacent coronal human brain sections were incubated with 30 pM [125I]PYY3–36 and in the presence of either 100 or 1000 nM BIIE0246. Non-specific binding was determined in the presence of 1 μM pNPY. See list of abbreviations for anatomical identification. Scale bar represents 20 mm.

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Table 4. Quantitative autoradiographic distribution of [125I]PYY3–36 binding sites in the marmoset monkey and human brainsThumbnail image of

The antagonistic properties of BIIE0246 were investigated next using various in vitro bioassays. In the rabbit saphenous vein and human cerebral arteries (two Y1 bioassays; Cadieux et al., 1993; Abounader et al., 1995), the Y2 antagonist (1 μM) had no agonistic properties by itself (data not shown) and failed to block NPY responses in those tissues (Table 5). In contrast, in the rat vas deferens (a prototypical pre-junctional Y2 bioassay; (Wahlestedt et al., 1986), increasing concentrations of BIIE0246 resulted in a parallel shift to the right of NPY concentration-response curves (Figure 6; Table 5) with a pA2 value of 8.1 as determined using a Schild plot (Table 5). Similarly, in the dog saphenous vein (a post-junctional Y2 bioassay; Pheng et al., 1997), BIIE0246 blocked the vasoconstriction induced by NPY with an apparent pA2 value of 8.6 (Table 5). In the rat colon (a Y2/Y4 bioassay; Pheng et al., 1999), 100 nM BIIE0246 induced a slight shift to the right of the NPY-induced contraction response curves (Figure 7, Table 5) while in the presence of 1 μM of the Y2 antagonist, the effect of NPY was markedly reduced, resulting in a decrease in the maximal response (Figure 7). Moreover, BIIE0246 (1 μM) was able to fully block the contractile effects induced by PYY3–36 but not those produced by [Leu31,Pro34]NPY and hPP (Figure 8).

Table 5. Comparative potencies of pNPY in the presence of various concentrations of BIIE0246 in several in vitro bioassaysThumbnail image of
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Figure 6. Concentration-response curves of pNPY in the presence and absence of various concentrations of BIIE0246 in the electrically stimulated rat vas deferens. Data represent the mean±s.e.mean of 4–8 determinations.

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Figure 7. Concentration-response curves of pNPY in the presence and absence of various concentrations of BIIE0246 in the isolated rat colon. Data represent the mean±s.e.mean of 4–6 determinations.

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Figure 8. Prototypical recording showing changes of tension (g) induced by PYY3–36, [Leu31,Pro34]NPY and hPP in the presence and absence of 1 μM BIIE0246 in the rat colon. W, represents washout.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. List of Abbreviations used in figures
  8. Acknowledgments
  9. References

We have demonstrated that BIIE0246 has high affinity for the Y2 receptor subtype while being inactive at the Y1, Y4 and Y5 subtypes in HEK 293 cells transfected with the respective receptor cDNA. In tissues containing heterogeneous population of NPY receptors such as the CNS, BIIE0246 was able to inhibit specific [125I]PYY3–36 binding sites with an affinity of 8–10 nM while failing to compete against [125I][Leu31,Pro34]PYY binding sites. Quantitative receptor autoradiographic studies demonstrated further that BIIE0246 competed for all specifically bound [125I]PYY3–36 labelling in most regions of the rat, marmoset monkey and human brains. However, few areas especially in the hippocampal formation, also revealed the existence of a small but significant proportion of [125I]PYY3–36/BIIE0246-insensitive sites possibly of the Y5 subtype (Dumont et al., 1998a,1998b). Additionally, the antagonistic property and selectivity of BIIE0246 for the Y2 receptor was confirmed using various in vitro bioassays. No agonistic or antagonistic activities of BIIE0246 were observed in isolated tissues in which NPY-induced effects are mediated by the activation of the Y1 and Y4 receptor subtypes. In contrast, BIIE0246 acted as a potent antagonist in the rat vas deferens and dog saphenous vein, two prototypical Y2in vitro bioassays (Pheng et al., 1997; Wahlestedt et al., 1986). Hence, these functional and radioligand binding data confirm that BIIE0246 is the first potent and selective Y2 receptor antagonist to be characterized and represents a useful tool to investigate the physiological role(s) of the Y2 subtype.

Most recently, Doods and collaborators from Boerhinger-Ingelheim in Germany briefly described the characterization of the first non-peptide Y2 receptor antagonist, BIIE0246 (Doods et al., 1999; 5th International NPY Meeting, Cayman Island, April 17–22, 1999). They reported that BIIE0246 antagonized the effects of NPY in the rat vas deferens with an apparent pA2 value of 7.8 while having no apparent affinity for the human and rat Y1, Y4 and Y5 receptor subtypes. Our results confirm and extend these preliminary results by demonstrating that BIIE0246 is a potent competitor for NPY receptors expressed in HEK 293 cells transfected with the rat Y2 receptor cDNA while being inactive in HEK 293 cells expressing the rat Y1, Y4 or Y5 receptor gene. Moreover, using BIIE0246 we were able to show that most but not all specific [125I]PYY3–36 binding sites observed in the rat, marmoset monkey and human brains are of the Y2 subtype. These data reveal the usefulness of BIIE0246 to clearly establish the genuine presence of the Y2 receptor subtype in tissues expressing heterogeneous populations of NPY receptors. Using in vitro bioassays, we have also demonstrated that BIIE0246 can block the activation of the Y2 receptor subtype without affecting the action of NPY or its homologues on the Y1 and Y4 receptors. This is especially evident in the rat colon, in which BIIE0246 (1 μM) abolished the contractile effects of PYY3–36 but not that induced by [Leu31,Pro34]NPY and hPP and only partly blocking that of NPY. These data clearly demonstrate the ability of BIIE0246 to discriminate between the Y2 vs Y1, Y4 and Y5 receptor subtypes.

Earlier on, other molecules were proposed to act as Y2 antagonists. In the rat femoral artery, an analogue of benextramine, N,N′-bis[6-[N-(2- naphthylmethyl)amino]hexyl]-N,N′-(1,6- hexanediyl)diguanidine tetrahydrochloride was reported to block the effect of the preferential Y2 agonist, NPY13–36, without altering the vasoconstriction induced by [Leu31,Pro34]NPY (Chaurasia et al., 1994). However, receptor binding assays in the rat brain homogenates failed to demonstrate the selectivity of this compound for Y2-like vs Y1-like receptors (Chaurasia et al., 1994) and we were unable to confirm its significant affinity for any NPY receptors (unpublished results). More recently, a peptidergic Y2 receptor antagonist, T4[NPY33–36]4 has been developed (Grouzmann et al., 1997). As shown here, this analogue weakly inhibits specific [125I]PYY3–36 binding sites in rat brain membrane preparations, confirming subsequent studies performed by Grouzmann (1998). Moreover, in vitro bioassays revealed that T4[NPY33–36]4 is only a weak Y2 antagonist (Pheng et al., 1999) while binding assays performed in HEK 293 cells transfected with the rat Y1, Y2, Y4 or Y5 receptor cDNA demonstrated its rather poor selectivity (Dumont et al., 1999b). Accordingly, BIIE0246 is by far more potent and selective than T4[NPY33–36]4 as an antagonist of the Y2 receptor subtype.

The functional role(s) of the Y2 receptor in the organism remains to be clearly established. However, some data have been reported supporting its involvement in a variety of NPY-induced CNS effects based on the pharmacological profile using various agonists, especially C-terminal fragments. For example, electrophysiological data suggest that NPY, by acting on presynaptic Y2 subtypes, can inhibit glutamatergic excitatory synaptic transmission in the hippocampus (Colmers, 1990; Colmers & Bleakman, 1994; McQuiston & Colmers, 1996; Qian et al., 1997) and hence suppress epileptiform activity (Klapstein & Colmers, 1997). On the other hand, it is not clear if the anticonvulsive activity of NPY is mediated by a Y2 receptor as suggested by Klapstein & Colmers (1997) or by a Y5-like subtype as proposed by Woldbye et al. (1997). The use of BIIE0246 and the newly developed Y5 receptor antagonist (Criscione et al., 1998) should be most helpful to establish the implication of the Y2 and/or the Y5 receptor subtypes in that regard.

The Y2 receptor subtype has also been suggested to modulate NPY-induced effects on circadian rhythms (Golombek et al., 1996; Huhman et al., 1996) and baroreceptor reflexes (Barraco et al., 1990; Narvaez et al., 1993). However, various NPY receptor subtypes may be involved in the modulation of circadian rhythms (Chen & van den Pol, 1996) and the purported ‘Y3’ receptor subtype has been proposed to be involved in modulating cardiorespiratory responses, (Glaum et al., 1997; Grundemar et al., 1991). The use of BIIE0246 should help to clarify if these effects of NPY and homologues are mediated solely by the Y2 subtype or by more than one population of NPY receptors as seen in the rat colon (Pheng et al., 1999). Furthermore, the existence of a PYY-preferring receptor has been suggested to explain the 3–5 fold differences in potency between PYY and NPY in rat crypt intestinal cells (Laburthe, 1991; Laburthe et al., 1986; Servin et al., 1989) and dog adipocytes (Castan et al., 1992). However, the pharmacological profile reported for the ‘PYY-preferring receptor subtype’ is most similar to that of the cloned Y2 receptor (Gehlert et al., 1996c; Gerald et al., 1995; Michel et al., 1998). The use of BIIE0246 should establish whether the so called ‘PYY-preferring receptor’ is in fact the Y2 receptor, or not. Finally, the Y2 receptor antagonist should be most helpful to clearly establish the role of the Y2 receptor subtype in various cardiovascular tissues as demonstrated in the dog saphenous vein (this study) and as suggested by others (Modin, 1994; Nilsson et al., 1996b). The use of selective antagonist(s) is more suitable to determine the exact nature of the receptor subtype involved in a given physiological response. This is especially evident for the NPY receptor family, since none of the agonists developed thus far are truly selective for one receptor subtype (Michel et al., 1998). Accordingly, BIIE0246 should prove most useful to establish the physiological and/or pathophysiological implication(s) of the Y2 receptor subtype.

In summary, we have demonstrated using several receptor binding assays and in vitro bioassays that BIIE0246 is a potent and selective Y2 receptor antagonist devoid of high affinity for the Y1, Y4 and Y5 subtypes. To our knowledge, BIIE0246 represents the first potent and selective tool to precisely establish the potential roles of the Y2 receptor in various tissues and to molecularly dissect features of agonist vs antagonist recognition sites on this receptor. The availability of BIIE0246, in addition to Y2 knockout mice (Naveilhan et al., 1999) should also prove critical to demonstrate the involvement of this subtype in a given effect induced by NPY and related peptides.

List of Abbreviations used in figures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. List of Abbreviations used in figures
  8. Acknowledgments
  9. References

I-VI, cortical layer 1-6; Acb, Accumbens nucleus; A Hy, anterior hypothalamic area; Amy, Amygdaloid complex; AOB, accessory olfactory bulb; AO, anterior olfactory nucleus; AP, area postrema; AV, anteroventral thalamic nucleus; BST, bed nucleus of the stria terminalis; CA1, field CA1 of hippocampus; CA3, field CA3 of hippocampus; Cau, caudate; Ce, cerebellum; Cg, cingulate cortex; Cl, claustrum; CM, central medial thalamic nucleus; CPu caudate putamen (striatum); Cx, cortex; DG, dentate gyrus; DLG, dorsolateral geniculate nucelus; EP1, external plexiform layer of the olfactory bulb; Fr, frontal cortex; GrA, granular cell layer of the accessory olfactory bulb; Hi, hippocampus; IO, inferior olive; LH, lateral hypothalamic area; LS, lateral septal nucleus; LSD, lateral septal nucleus, dorsal part; LSI, lateral nucleus, intermediate part; MD, mediodorsal thalamic nucleus; MM, medial mammillary nucleus, medial part; MPS, medial preoptic area; NS, non-specific binding; Or, oriens layer of the hippocampus; Par, Parietal cortex; Pir, piriform cortex; Po, pontine nucleus; Put, putamen; Py, pyramidal cell layer of the hippocampus; Rad, stratum radiatum of the hippocampus; SNC, substantia nigra, compact part; SNL, substantia nigra, lateral part; SNR, substantia nigra, reticular part; Sol, nucleus of the solitary tract; Th, thalamus; Tu, olfactory tubercle; vHi, ventral part of the hippocampus; VHy, ventral part of the hypothalamus; VP, ventral posterior thalamic nucleus; VTA, ventral tegmental area

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. List of Abbreviations used in figures
  8. Acknowledgments
  9. References

This study was supported by the Medical Research Council of Canada. A. Cadieux, E. Hamel and R. Quirion are ‘Chercheur-Boursiers’ of the ‘Fonds de la recherche en Santé du Québec’. L.H. Pheng has a studentship from the Heart and Stroke Foundation of Canada and D. Regoli has salary support from the Medical Research Council of Canada. The authors would like to thank Mrs Danielle Cécyre, Coordinator of the Douglas Hospital Research Centre Brain Bank, for supplying us with human brain tissues.

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  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. List of Abbreviations used in figures
  8. Acknowledgments
  9. References
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