Determination of affinity and activity of ligands at the human neuropeptide Y Y4 receptor by flow cytometry and aequorin luminescence

Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y1, Y2, and Y5 receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y4 receptor (hY4R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K(4)]hPP has high affinity (Kd 5.6 nM) to the hY4R, it was used as a probe in a flow cytometric binding assay. Specific binding of cy5-[K(4)]hPP to hY4R was visualized by confocal microscopy. The hY(4)R, the chimeric G protein G(qi5) and mitochondrially targeted apoaequorin were stably coexpressed in CHO cells. Aequorin luminescence was quantified in a microplate reader and by a CCD camera. By application of these methods 3-cyclohexyl-N-[(3-1H-imidazol-4-ylpropylamino)(imino)methyl]propanamide (UR-AK49) was discovered as the first nonpeptidic Y4R antagonist (pKi 4.17), a lead to be optimized in terms of potency and selectivity.     

Novel Cell Line Selectively Expressing Neuropeptide Y‐Y2 Receptors

Abstract

Neuropeptide Y (NPY) recognition by the human neuroblastoma cell lines SiMa, Kelly, SH‐SY5Y, CHP‐234, and MHH‐NB‐11 was analyzed in radioactive binding assays using tritiated NPY. For the cell lines CHP‐234 and MHH‐NB‐11 binding of [³H]propionyl‐NPY was observed with K_d‐values of 0.64 ± 0.07 nM and 0.53 ± 0.12 nM, respectively, determined by saturation analysis with non‐linear regression. The receptor subtype was determined by competition analysis using the subtype selective NPY analogues [Leu³¹, Pro³⁴]‐NPY (NPY‐Y₁, NPY‐Y₅), [Ahx^5‐24]‐NPY (NPY‐Y₂), [Ala³¹, Aib³²]‐NPY (NPY‐Y₅), NPY [3‐36] (NPY‐Y₂, NPY‐Y₅), and NPY [13‐36] (NPY‐Y₂). Both cell lines, CHP‐234 and MHH‐NB‐11, the latter one being characterized for NPY receptors for the first time, showed exclusive expression of NPY‐Y₂ receptors. In both cell lines binding of NPY induced signal transduction, which was monitored as reduction of forskolin‐induced cAMP production in an ELISA.     

Pancreatic Polypeptide Is Recognized by Two Hydrophobic Domains of the Human Y4 Receptor Binding Pocket

Structural characterization of the human Y₄ receptor (hY₄R) interaction with human pancreatic polypeptide (hPP) is crucial, not only for understanding its biological function but also for testing treatment strategies for obesity that target this interaction. Here, the interaction of receptor mutants with pancreatic polypeptide analogs was studied through double-cycle mutagenesis. To guide mutagenesis and interpret results, a three-dimensional comparative model of the hY₄R-hPP complex was constructed based on all available class A G protein-coupled receptor crystal structures and refined using experimental data. Our study reveals that residues of the hPP and the hY₄R form a complex network consisting of ionic interactions, hydrophobic interactions, and hydrogen binding. Residues Tyr^2.64, Asp^2.68, Asn^6.55, Asn^7.32, and Phe^7.35 of Y₄R are found to be important in receptor activation by hPP. Specifically, Tyr^2.64 interacts with Tyr²⁷ of hPP through hydrophobic contacts. Asn^7.32 is affected by modifications on position Arg³³ of hPP, suggesting a hydrogen bond between these two residues. Likewise, we find that Phe^7.35 is affected by modifications of hPP at positions 33 and 36, indicating interactions between these three amino acids. Taken together, we demonstrate that the top of transmembrane helix 2 (TM2) and the top of transmembrane helices 6 and 7 (TM6–TM7) form the core of the peptide binding pocket. These findings will contribute to the rational design of ligands that bind the receptor more effectively to produce an enhanced agonistic or antagonistic effect.     

Molecular Modeling of the NPY Binding Site on the Y1 Receptor

A three-dimensional model of the neuropeptide Y (NPY) - rat Y₁ (rY₁) receptor complex and of the NPY _13-36 - rY₁ receptor complex was constructed by molecular modeling based on the electron density projection map of rhodopsin and on site-directed mutagenesis studies of neuropeptide receptors. In order to further guide the modeling, the nucleotide sequences encoding Trp287, Cys295 and His297 in the third extracellular loop of the rY₁ receptor, were altered by site-directed mutagenesis experiments. Single-point mutated receptors were expressed in COS-7 cells, and tested for their ability to bind radio labelled NPY (³H-NPY). Mutations of Trp287 and His297 completely abolished binding of ³H-NPY. The Cys295Ser mutation only slightly decreased the binding of ³H-NPY, suggesting that the involvement of Cys295 in a disulphide bond is not essential for maintaining the correct three-dimensional structure of the binding site for NPY. Molecular dynamics simulations of NPY-rY₁ receptor interactions suggested that Asp199, Asp103 and Asp286 in the receptor interact, respectively, with Lys4, Arg33 and Arg35 of NPY. The simulations also suggested that His297 acts as a hydrogen acceptor from Arg35 in NPY, and that Tyr1 of NPY interacts with a binding pocket on the receptor formed by Asn115, Asp286, Trp287 and His297. Tyr36 in NPY interacted both with Thr41 and Tyr99 via hydrogen bonds, and also with Asn296, His297 and Phe301. The present study suggests that amino acid residues at the extracellular end of the transmembrane helices and in the extracellular loops are strongly involved in binding to NPY and NPY_13-36.Electronic Supplementary Material available.     

Molecular characterization of the ligand–receptor interaction of the neuropeptide Y family

Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) belong to the NPY hormone family and activate a class of receptors called the Y‐receptors, and also belong to the large superfamily of the G‐protein coupled receptors. Structure–affinity and structure–activity relationship studies of peptide analogs, combined with studies based on site‐directed mutagenesis and anti‐receptor antibodies, have given insight into the individual characterization of each receptor subtype relative to its interaction with the ligand, as well as to its biological function. A number of selective antagonists at the Y₁‐receptor are available whose structures resemble that of the C‐terminus of NPY. Some of these compounds, like BIBP3226, BIBO3304 and GW1229, have recently been used for in vivo investigations of the NPY‐induced increase in food intake. Y₂‐receptor selective agonists are the analog cyclo‐(28/32)‐Ac‐[Lys²⁸‐Glu³²]‐(25–36)‐pNPY and the TASP molecule containing two units of the NPY segment 21–36. Now the first antagonist with nanomolar affinity for the Y₂‐receptor is also known, BIIE0246. So far, the native peptide PP has been shown to be the most potent ligand at the Y₄‐receptor. However, by the design of PP/NPY chimera, some analogs have been found that bind not only to the Y₄‐, but also to the Y₅‐receptor with subnanomolar affinities, and are as potent as NPY at the Y₁‐receptor. For the characterization of the Y₅‐receptor in vitro and in vivo, a new class of highly selective agonists is now available. This consists of analogs of NPY and of PP/NPY chimera which all contain the motif Ala³¹‐Aib³². This motif has been shown to induce a 3₁₀‐helical turn in the region 28–31 of NPY and is suggested to be the key motif for high Y₅‐receptor selectivity. The results of feeding experiments in rats treated with the first highly specific Y₅‐receptor agonists support the hypothesis that this receptor plays a role in the NPY‐induced stimulation of food intake. In conclusion, the selective compounds for the different Y‐receptor subtypes known so far are promising tools for a better understanding of the physiological properties of the hormones of the NPY family and related receptors. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.     

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y₁₂ receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y₁ and P2Y₁₂. We used the [³H]PSB-0413 selective P2Y₁₂ receptor antagonist radioligand to reevaluate the number of P2Y₁₂ receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [³H]PSB-0413 bound to 425 ± 50 sites/platelet (K_D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y₁₂, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y₁ ligand MRS2179 and the P2X₁ ligand α,β-Met-ATP did not displace [³H]PSB-0413 binding. Patients with severe P2Y₁₂ deficiency displayed virtually no binding of [³H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y₁₂ receptor had normal binding. Studies in mice showed that: (1) [³H]PSB-0413 bound to 634 ± 87 sites/platelet (K_D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [³H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y₁₂ receptors, to identify patients with P2Y₁₂ deficiencies or quantify the effect of P2Y₁₂ targeting drugs.     

NG-Acyl-argininamides as NPY Y1 receptor antagonists: Influence of structurally diverse acyl substituents on stability and affinity

N^G-Acylated argininamides, covering a broad range of lipophilicity (calculated log D values: ?1.8–12.5), were synthesized and investigated for NPY Y₁ receptor (Y₁R) antagonism, Y₁R affinity and stability in buffer (N^G-deacylation, yielding BIBP 3226). Broad structural variation of substituents was tolerated. The K_i (binding) and K_b values (Y₁R antagonism) varied from low nM to one-digit μM. Most of the compounds proved to be sufficiently stable at pH 7.4 over 90 min to determine reliable pharmacological data in vitro. Exceptionally high instability was detected when a succinyl moiety was attached to the guanidine, probably, due to an intramolecular cleavage mechanism.     

P2Y2 receptor agonist with enhanced stability protects the heart from ischemic damage in vitro and in vivo

Extracellular nucleotides acting via P2 receptors play important roles in cardiovascular physiology/pathophysiology. Pyrimidine nucleotides activate four G protein-coupled P2Y receptors (P2YRs): P2Y₂ and P2Y₄ (UTP-activated), P2Y₆, and P2Y₁₄. Previously, we showed that uridine 5′-triphosphate (UTP) activating P2Y₂R reduced infarct size and improved mouse heart function after myocardial infarct (MI). Here, we examined the cardioprotective role of P2Y₂R in vitro and in vivo following MI using uridine-5′-tetraphosphate δ-phenyl ester tetrasodium salt (MRS2768), a selective and more stable P2Y₂R agonist. Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y₂R antagonist, AR-C118925 (5-((5-(2,8-dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide). In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24 h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1 % vs. 33.4 ± 2.7 %, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y₂R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

Modeling of Neuropeptide Receptors Y1, Y4, Y5, and Docking Studies with Neuropeptide Antagonist Analogues: Implications for Selectivity

Abstract

Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y₁-Y₅ and y₆. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Dimeric argininamide-type neuropeptide Y receptor antagonists: Chiral discrimination between Y1 and Y4 receptors

The structurally related peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are endogenous agonists of the NPY receptor (YR) family, which in humans comprises four functionally expressed subtypes, designated Y₁R, Y₂R, Y₄R and Y₅R. Nonpeptide antagonists with high affinity and selectivity have been described for the Y₁R, Y₂R and Y₅R, but such compounds are still lacking for the Y₄R. In this work, the structures of the high affinity selective (R)-argininamide-type Y₁R antagonists BIBP3226 and BIBO3304 were linked via the guanidine or urea moieties to give homo-dimeric argininamides with linker lengths ranging from 31 to 41 atoms. Interestingly, the twin compounds proved to be by far less selective for the Y₁R than the R-configured monovalent parent compounds. The decrease in selectivity ratio was most pronounced for Y₁R versus Y₄R subtype, resulting in comparable affinities of bivalent ligands for Y₁R and Y₄R (e.g. UR-MK177 ((R,R)-49): K_i = 230 nM (Y₁R) and 290 nM (Y₄R)). With a K_i value of 130 nM and a K_b value of 20 nM, UR-MK188 ((R,R)-51) was superior to all Y₄R antagonists known to date. The S,S-configured optical antipodes of UR-MK177 and UR-MK188 (UR-MEK381 ((S,S)-49) and UR-MEK388 ((S,S)-51)) were synthesized to investigate the stereochemical discrimination by the different receptor subtypes. Whereas preference for R,R-configured argininamides was characteristic of the Y₁R, stereochemical discrimination by the Y₄R was not observed. This may pave the way to selective Y₄R antagonists.     

Synthesis and preliminary evaluation of [H]PSB-0413, a selective antagonist radioligand for platelet P2Y12 receptors

The selective antagonist radioligand [³H]2-propylthioadenosine-5′-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([³H]PSB-0413) was prepared by catalytic hydrogenation of its propargyl precursor with a high specific radioactivity of 74 Ci/mmol. In preliminary saturation binding studies, [³H]PSB-0413 showed high affinity for platelet P2Y₁₂ receptors with a K_D value of 4.57 nM. Human platelets had a high density of P2Y₁₂ receptors exhibiting a B_max value of 7.66 pmol/mg of protein.     

UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation

Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′-triphosphate (UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the most potent agonist among several nucleotides tested. Adenosine 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also potent agonists, characteristic of the P2Y₂ or P2Y₄ receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2 phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is mediated by a P2Y₂ or P2Y₄ receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR.     

[Lys(DOTA)4]BVD15, a novel and potent neuropeptide Y analog designed for Y1 receptor-targeted breast tumor imaging

We substituted a truncated neuropeptide Y (NPY) analog, [Pro³⁰, Tyr³², Leu³⁴]NPY(28-36)NH₂ also called BVD15, at various positions with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7-10-tetraacetic acid) and evaluated the effect of the coupling position with the binding affinity for NPY Y₁ receptors (NPY1R). Our data suggest that [Lys(DOTA)⁴]BVD15 (K_i = 63 ± 25 nM vs. K_i = 39 ± 34 nM for BVD15) is a potent NPY analog suitable for radiolabeling with metallo positron emitters for PET imaging of breast cancer.     

Regional expression of P2Y4 receptors in the rat central nervous system

P2Y receptors are G protein-coupled receptors composed of eight known subunits (P2Y_{1, 2, 4, 6, 11, 12, 13, 14}), which are involved in different functions in neural tissue. The present study investigates the expression pattern of P2Y₄ receptors in the rat central nervous system (CNS) using immunohistochemistry and in situ hybridization. The specificity of the immunostaining has been verified by preabsorption, Western blot, and combined use of immunohistochemistry and in situ hybridization. Neurons expressing P2Y₄ receptors were distributed widely in the rat CNS. Heavy P2Y₄ receptor immunostaining was observed in the magnocellular neuroendocrine neurons of the hypothalamus, red nucleus, pontine nuclei, mesencephalic trigeminal nucleus, motor trigeminal nucleus, ambiguous nucleus, inferior olive, hypoglossal nucleus, and dorsal motor vagus nucleus. Both neurons and astrocytes express P2Y₄ receptors. P2Y₄ receptor immunostaining signals were mainly confined to cell bodies and dendrites of neurons, suggesting that P2Y₄ receptors are mainly involved in regulating postsynaptic events. In the hypothalamus, all the vasopressin (VP) and oxytocin (OT) neurons and all the orexin A neurons were immunoreactive for P2Y₄ receptors. All the neurons expressing P2Y₄ receptors were found to express N-methyl-d-aspartate receptor 1 (NR1). These data suggest that purines and pyrimidines might be involved in regulation of the release of the neuropeptides VP, OT, and orexin in the rat hypothalamus via P2Y₄ receptors. Further, the physiological and pathophysiological functions of the neurons may operate through coupling between P2Y₄ receptors and NR1.     

NEUROPEPTIDE Y RECEPTORS FROM CALF BRAIN: EFFECT OF CRUDE CONUS VENOM PREPARATIONS ON [H]NPY BINDING

NPY receptors are identified in calf frontal cortex and hippocampus membrane preparations by binding of N-[propionyl-³H] neuropeptide Y. Saturation and competition binding data with PYY, NPY-(18–36) and NPY itself fit with a single class of sites: for the radioligand K_D = 1.4 ± 0.5 nM, B_max = 434 ± 180 fmol/mg protein in frontal cortex, K_D = 0.7 ± 0.2 nM, B_max = 267 ± 50 fmol/mg protein in hippocampus. Competition curves of the Y₁-subtype selective agonist [Leu³¹, Pro³⁴]NPY are biphasic in both membrane preparations: high affinity sites (i.e. Y₁-subtype) amount to 80% in frontal cortex and 23% in hippocampus. The remaining sites are of the Y₂-subtype. Out of 23 Conus venom preparations, 17 inhibit the binding of [³H]NPY in both membrane preparations, but only two of them (from Conus aulicus and C. pennaceus) do so with high potency (ic₅₀ < 5 μg protein/ml). Only one venom preparation (from C. mercator) had weak discriminatory properties (ic₅₀Y₂/ic₅₀Y₁ = 6). Venom from C. anemone increased the [³H]NPY binding 5-fold and with an ic₅₀ of 15–18 μg protein/ml. This binding occurred to the venom itself and was unrelated to the NPY receptors since it was equally potent when displaced by [Leu³¹, Pro³⁴]NPY, NPY-(18–36), PYY and NPY. Copyright © 1996 Elsevier Science Ltd     

Conformational Analysis of Neuropeptide Y Segments by CD,NMR Spectroscopy and Restrained Molecular Dynamics

Neuropeptide Y (NPY), a peptide amide comprising 36 residue has been shown to act as a potent vasoconstrictor. In order to shed light on the structural requirements for the biological activities with respect to the different prerequisites for affinity to the NPY receptor subtypes Y₁ and Y₂, in the present study the syntheses and conformational analyses of two C-terminal segments, NPY(18–36) and NPY(13–36), are described. The results obtained by CD measurements, two-dimensional NMR spectros copy and a conformational refinement of the NMR-derived structure by molecular mechanics simulations support the findings of previously published structure –activity relationship studies for biologically active and selective compounds. In particular, the α-helical conformation as well as an appropriate exposure of the side chains of the critical C-terminal dipeptide within NPY(18–36) are in agreement with the prerequisites proposed for Y₂ receptor binding of that segment.     

First selective agonist of the neuropeptide Y1‐receptor with reduced size

Selective NPY analogues are potent tools for tumour targeting. Their Y₁‐receptors are significantly over‐expressed in human breast tumours, whereas normal breast tissue only expresses Y₂‐receptors. The endogenous peptide consists of 36 amino acids, whereas smaller peptides are preferred because of better labelling efficiencies. As Y₁‐receptor agonists enhance the tumour to background ratio compared to Y₁‐receptor antagonists, we were interested in the development of Y₁‐receptor selective agonists. We designed 19 peptides containing the C‐terminus of NPY (28–36) with several modifications. By using competition receptor binding affinity assays, we identified three NPY analogues with high Y₁‐receptor affinity and selectivity. Metabolic stability studies in human blood plasma of the N‐terminally 5(6)‐carboxyfluorescein (CF) labelled peptides resulted in half‐lives of several hours. Furthermore, the degradation pattern revealed proteolytic degradation of the peptides by amino peptidases. The most promising peptide was further investigated in receptor activation and internalization studies. Signal transduction assays revealed clear agonistic properties, which could be confirmed by microscopy studies that showed clear Y₁‐receptor internalization. For the first time, here we show the design and characterization of a small Y₁‐receptor selective agonist. This agonist might be a useful novel ligand for NPY‐mediated tumour diagnostics and therapeutics. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Neuropeptide Y receptors: ligand binding and trafficking suggest novel approaches in drug development

NPY, PYY and PP constitute the so‐called NPY hormone family, which exert its biological functions in humans through YRs (Y₁, Y₂, Y₄ and Y₅). Systematic modulation of YR function became important as this multireceptor/multiligand system is known to mediate various essential physiological key functions and is involved in a variety of major human diseases such as epilepsy, obesity and cancer. As several YRs have been found to be overexpressed on different types of malignant tumors they emerge as promising target in modern drug development. Here, we summarize the current understanding of YRs function and the molecular mechanisms of ligand binding and trafficking. We further address recent advances in YR‐based drug design, the development of promising future drug candidates and novel approaches in YR‐targeted tumor diagnostics and therapy opportunities. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Distribution of P2Y<Subscript>2</Subscript> receptors in the guinea pig enteric nervous system and its coexistence with P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> receptors,neuropeptide Y,nitric oxide synthase and calretinin

The distribution of P2Y₂ receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y₂ with P2X₂ and P2X₃ receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y₂-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y₂-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y₂-ir neurons could be Dogiel type 1. About 40–60% P2X₃-ir neurons were immunoreactive for P2Y₂ in the myenteric plexus and all the P2X₃-ir neurons expressed the P2Y₂ receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y₂, especially in the myenteric plexus of the small intestine; no P2Y₂-ir neurons were immunoreactive for P2X₂ receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y₂-ir and P2X₃-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.