Pancreatic polypeptide receptors: affinity, sodium sensitivity and stability of agonist binding

Cloned rat, human and guinea-pig Y4 pancreatic polypeptide (PP) receptors expressed in Chinese hamster ovary (CHO) cells, as well as the rabbit Y4-like PP receptor, show a selective sensitivity to Na+ over K+ ion in PP attachment, but little sensitivity to Na+ in dissociation of bound PP peptides. Agonist binding to Y4 receptors of intact CHO cells also shows much greater sensitivity to Na+ over K+, and a tenacious attachment of the bound agonist. Binding sensitivity to K+ is greatly enhanced upon receptor solubilization. Pancreatic polypeptide sites also show large sensitivity to modulators of Na+ transport such as N5-substituted amilorides and to RFamides, as different from Y1 or Y2 receptors. Thus, PP binding is modulated by cation-induced changes in site environment (with selectivity for Na+) and ultimately results in a blocking attachment. This would support receptor operation in the presence of ion gradients, as well as prolonged agonist-delimited signaling activity (which can include partial antagonism). Also, this could point to an evolutionary adaptation enabling small numbers of PP receptors to perform extensive metabolic tasks in response to low agonist signals.     

Self-regulation of agonist activity at the Y receptors

Neuropeptide Y (NPY) is one of the most abundant neuropeptides, and is likely to be present at nanomolar levels over extended periods in the synaptic space of many forebrain areas. This might be linked to an evolved generalized toning activity through a number of other peptide receptors that use C-terminally amidated agonists (with LHRH and orexin receptors and GIR as examples). However, the Y1 and Y2 receptors (which constitute the bulk of Y receptors active in the neural matrix) possess subnanomolar affinities that, at saturating NPY levels, could produce excessive signaling, as well as receptor losses via repeated endocytosis. The related Y4 receptor shows an even higher agonist affinity, and faces the same problem in visceral and neural locations accessible to pancreatic polypeptide (PP). An examination of agonist peptide interaction with Y receptors shows that Y1 and Y4 receptors in particular (as located on either the intact cells, or on particulates derived from various cell types) develop a blockade dependent on ligand concentration, with the blocking ranks of [NPY]>[peptide YY] (PYY) for the Y1, and [human PP]>[PYY-related Y4 agonist] for the Y4 receptor. This blockade is also echoed in a concentration-related reduction in biological activity of primary agonists (NPY and PP), resembling a partial agonism, and is influenced especially by the allosteric interactivity of agonists. With the Y2 receptor, the blocking by agonists is less pronounced, but the signaling by NPY-related peptides is apparently less than with PYY-related agonists. The extended occupancy and self-attenuation of primary agonist activity at Y receptors could represent an evolutionary solution contributing to a balancing of metabolic signaling, agonist clearance and receptor conservation.     

Recent developments in our understanding of the physiological role of PP-fold peptide receptor subtypes

The three peptides pancreatic polypeptide (PP), peptide YY (PYY), and neuropeptide Y (NPY) share a similar structure known as the PP-fold. There are four known human G-protein coupled receptors for the PP-fold peptides, namely Y1, Y2, Y4, and Y5, each of them being able to bind at least two of the three endogenous ligands. All three peptides are found in the circulation acting as hormones. Although NPY is only released from neurons, PYY and PP are primarily found in endocrine cells in the gut, where they exert such effects as inhibition of gall bladder secretion, gut motility, and pancreatic secretion. However, when PYY is administered in an experimental setting to animals, cloned receptors, or tissue preparations, it can mimic the effects of NPY in essentially all studies, making it difficult to study the effects of PP-fold peptides and to delineate what receptor and peptide accounts for a particular effect. Initial studies with transgenic animals confirmed the well-established action of NPY on metabolism, food-intake, vascular systems, memory, mood, neuronal excitability, and reproduction. More recently, using transgenic techniques and novel antagonists for the Y1, Y2, and Y5 receptors, NPY has been found to be a key player in the regulation of ethanol consumption and neuronal development.     

Spinal and peripheral modulation of gastric acid secretion and arterial pressure by neuropeptide Y, peptide YY, and pancreatic polypeptide in rats

Spinal and peripheral modulation of pentagastrin-stimulated gastric acid secretion by the pancreatic polypeptide-fold (PP-fold) peptides, neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP), in urethane-anesthetized rats was evaluated. Neuropeptide Y, PYY, and PP (400 pmol) were administered via intravenous (IV) and intrathecal (IT) injections. The ₂ antagonist, yohimbine, was used to evaluate the role of the ₂ adrenergic receptors in the modulation of pentagastrin-stimulated gastric acid secretion by NPY, PYY, and PP. Peptide YY and PP (IV) rapidly increased pentagastrin-stimulated gastric acid secretion. Peptide YY and PP (IT) increased pentagastrin-stimulated gastric acid secretion following administration into the thoracic (T8–T10) region of the spinal cord. The ₂ adrenergic receptor antagonist, yohimbine, did not modify the increases in pentagastrin-stimulated gastric acid secretion following PYY and PP (IV or IT) administration. Neuropeptide Y (IT) decreased pentagastrin-stimulated gastric acid secretion. However, in the presence of ₂ adrenergic receptor blockade, pentagastrin-stimulated gastric acid secretion was potentiated by NPY (IT) administration. Therefore, the inhibitory effect of NPY (IT) on pentagastrin-stimulated gastric acid secretion required the activation of ₂ adrenergic receptors in the spinal cord of rats. Mean arterial blood pressure (MAP) was increased immediately following NPY and PYY (IV) administration. During the same time period, PP (IV) decreased MAP in anesthetized rats. Mean arterial blood pressure was rapidly increased by NPY and PYY (IT) in anesthetized rats. The increase in MAP following PYY (IT) was partially attenuated in the presence of yohimbine. The modulation of MAP and gastric acid secretion by the PP-fold peptides occurred by independent mechanisms at spinal and peripheral sites in the rat. The modulation of pentagastrin-stimulated gastric acid secretion by PYY and PP in rats differed from that of the third member of the PP-fold family, NPY, following spinal and peripheral administration.     

Y-receptor affinity modulation by the design of pancreatic polypeptide/neuropeptide Y chimera led to Y(5)-receptor ligands with picomolar affinity

Neuropeptide Y (NPY) and pancreatic polypeptide (PP) bind to the Y-receptors with very different affinities: NPY has high affinity for the receptors Y(1), Y(2) and Y(5), while PP binds only to Y(4)-receptor with picomolar affinity. By exchanging of specific amino acid positions between the two peptides, we developed 38 full-length PP/NPY chimeras with binding properties that are completely different from those of the two native ligands. Pig NPY (pNPY) analogs containing the segment 19-23 from human PP (hPP) bound to the Y-receptors with much lower affinity than NPY itself. The affinity of the hPP analog containing the pNPY segments 1-7 and 19-23 was comparable to that of pNPY at the Y(1)- and Y(5)-receptor subtypes, and to that of hPP at the Y(4)-receptor. Furthermore, the presence of the segments 1-7 from chicken PP (cPP) and 19-23 from pNPY within the hPP sequence led to a ligand with IC(50) of 40 pM at the Y(5)-receptor. This is the most potent Y(5)-receptor ligand known so far, with 15-fold higher affinity than NPY.     

The antiparallel pancreatic polypeptide fold in the binding of neuropeptide Y to Y1 and Y2 receptors

Neuropeptide Y (NPY) belongs to the pancreatic polypeptide fold (PP-fold) family of regulatory peptides. Analysis of circular dicroic spectra of NPY showed that it has a high degree of secondary structure in aqueous solution which is in agreement with the globular, folded crystal structure of PP. Using three different approaches with synthetic peptides, we have probed the importance of the PP-fold structure in the interaction of NPY with two types of binding sites, Y1 and Y2 receptors. First, stepwise construction of the NPY molecule from the C-terminal amidated end, showed that although C-terminal fragments encompassing most of the long alpha-helix reacted reasonably well with the Y2 receptor, both Y1 and Y2 receptors required the presence of both ends of the PP-fold for full activity. Second, perturbation of the PP-fold by substitution with a helix-breaking proline residue, resulted in the loss of recognition of the N-terminal segment of the molecule by both types of receptors. Finally, a hybrid analog was constructed in which the essential, but by itself inactive, C-terminal segment of NPY was joined with the PP-fold motif of PP. This segment of PP is only 43% homologous to the similar motif in NPY, and most of the common residues cluster in the hydrophobic core of the fold. Nevertheless, the hybrid analog reacted with almost full potency on the Y2 receptors. It is concluded that the antiparallel PP-fold is of structural importance for the receptor binding of NPY, and that its main function is to present the combined C- and N-terminal segments of the molecule to the receptors.     

Regulation of Neuropeptide Y Release by Neuropeptide Y Receptor Ligands and Calcium Channel Antagonists in Hypothalamic Slices

Neuropeptide Y (NPY) is an important regulator of energy balance in mammals through its orexigenic, antithermogenic, and insulin secretagogue actions. We investigated the regulation of endogenous NPY release from rat hypothalamic slices by NPY receptor ligands and calcium channel antagonists. High-potassium stimulation (60 mM) of the slices produced a calcium-dependent threefold increase in NPY release above basal release. The Y2 receptor agonists NPY(13-36) and N-acetyl[Leu28,Leu31]NPY(24-36), the Y4 agonist rat pancreatic polypeptide (rPP), and the Y4/Y5 agonist human pancreatic polypeptide (hPP) significantly reduced both basal and stimulated NPY release. NPY(13-36)-induced reduction of NPY release could be partially prevented in the presence of the weak Y2 antagonist T4-[NPY(33-36)]4, whereas the hPP- and rPP-induced inhibition of release was not affected by the Y5 antagonist CGP71683A or the Y1 antagonist BIBP3226. The selective Y1, Y2, and Y5 antagonists had no effect on either basal or potassium-stimulated release when administered alone. The calcium channel inhibitors omega-conotoxin GVIA (N-type), omega-agatoxin TK (P/Q-type), and omega-conotoxin MVIIC (Q-type) all significantly inhibited potassium-stimulated NPY release, without any effect on basal release, whereas nifedipine had no effect on either basal or stimulated release. Addition of both omega-conotoxin GVIA and omega-agatoxin TK together completely inhibited the potassium-stimulated release. In conclusion, we have demonstrated that NPY release from hypothalamic slices is calcium-dependent, involving N-, P-, and Q-type calcium channels. NPY release is also inhibited by Y2 agonists and rPP/hPP, suggesting that Y2 and Y4 receptors may act as autoreceptors on NPY-containing nerve terminals.     

Receptor subtype-specific docking of Asp6.59 with C-terminal arginine residues in Y receptor ligands

Y receptors (YRs) are G protein-coupled receptors whose Y(1)R, Y(2)R, and Y(5)R subtypes preferentially bind neuropeptide Y (NPY) and peptide YY, whereas mammalian Y(4)Rs show a higher affinity for pancreatic polypeptide (PP). Comparison of YR orthologs and paralogs revealed Asp(6.59) to be fully conserved throughout all of the YRs reported so far. By replacing this conserved aspartic acid residue with alanine, asparagine, glutamate, and arginine, we now show that this residue plays a crucial role in binding and signal transduction of NPY/PP at all YRs. Sensitivity to distinct replacements is, however, receptor subtype-specific. Next, we performed a complementary mutagenesis approach to identify the contact site of the ligand. Surprisingly, this conserved residue interacts with two different ligand arginine residues by ionic interactions; although in Y(2)R and Y(5)R, Arg(33) is the binding partner of Asp(6.59), in Y(1)R and Y(4)R, Arg(35) of human PP and NPY interacts with Asp(6.59). Furthermore, Arg(25) of PP and NPY is involved in ligand binding only at Y(2)R and Y(5)R. This suggests significant differences in the docking of YR ligands between Y(1/4)R and Y(2/5)R and provides new insights into the molecular binding mode of peptide agonists at GPCRs. Furthermore, the proposed model of a subtype-specific binding mode is in agreement with the evolution of YRs.     

Neuropeptide Y receptor in cultured vascular smooth muscle cells: ligand binding and increase in cytosolic free Ca2+

We have previously shown that neuropeptide Y (NPY) increases cytosolic free Ca2+ concentration [( Ca2+]i) in porcine aortic smooth muscle cells. In this study, specific NPY receptor binding sites were identified in the cells by use of [125I]Bolton-Hunter NPY [( 125I]BH-NPY). Binding was to a single population of the sites with a Kd of 1.1 +/- 0.2 nM and a Bmax of 0.68 +/- 0.10 pmol/mg protein. [125I]BH-NPY binding was displaced by NPY-related peptides including members of the pancreatic polypeptide (PP) family. The potency of these peptides other than human PP for displacing [125I]BH-NPY binding was substantially consistent with their potency for increasing [Ca2+]i. Human PP had no effect on [Ca2+]i even at 10(-5) M, but it inhibited the NPY-induced increase in [Ca2+]i with a potency comparable to that for displacing [125I]BH-NPY binding. NPY(13-36) was about 500 and 300 times less effective than porcine NPY in increasing [Ca2+]i and in displacing [125I]BH-NPY binding, respectively, showing that the NPY receptor in cultured vascular smooth muscle cells is of the Y1-type.     

Strongly altered receptor binding properties in PP and NPY chimeras are accompanied by changes in structure and membrane binding

Neuropeptide Y (NPY) and the pancreatic polypeptide (PP) are members of the neuropeptide Y family of hormones. They bind to the Y receptors with very different affinities: Whereas PP is highly selective for the Y(4) receptor, NPY displays highest affinites for Y(1), Y(2), and Y(5) receptor subtypes. Introducing the NPY segment 19-23 into PP leads to an increase in affinity at the Y(1) and Y(2) receptor subtypes whereas the exchange of this segment from PP into NPY leads to a large decrease in affinity at all receptor subtypes. PP displays a very stable structure in solution, with the N terminus being back-folded onto the C-terminal alpha-helix (the so-called PP-fold). The helix of NPY is less stable and the N terminus is freely diffusing in solution. The exchange of this segment, however, does not alter the PP-fold propensities of the chimeric peptides in solution. The structures of the phospholipid micelle-bound peptides serving to mimic the membrane-bound species display segregation into a more flexible N-terminal region and a well-defined alpha-helical region. The introduction of the [19-23]-pNPY segment into hPP leads to an N-terminal extension of the alpha-helix, now starting at Pro(14) instead of Met(17). In contrast, a truncated helix is observed in [(19)(-)(23)hPP]-pNPY, starting at Leu(17) instead of Ala(14). All peptides display moderate binding affinities to neutral membranes (K(assoc) in the range of 1.7 to 6.8 x 10(4) mol(-)(1) as determined by surface plasmon resonance) with the differences in binding being most probably related to the exchange of Arg-19 (pNPY) by Glu-23 (hPP). Differences in receptor binding properties between the chimeras and their parental peptides are therefore most likely due to changes in the conformation of the micelle-bound peptides.     

FMRFamides exert a unique modulation of rodent pancreatic polypeptide sensitive neuropeptide Y (NPY) receptors

FMRFamide and related peptides (RFamides) were found to inhibit the association binding of iodinated human pancreatic polypeptide ([125I]hPP) to Y5-like neuropeptide Y (NPY) receptor in rodent tissues. An allosteric regulation of the activity of the rodent kidney PP-sensitive neuropeptide Y (NPY) receptor by RFamides was indicated by potency decrease with particle concentration in the inhibition of the association binding of 125I-labeled human pancreatic polypeptide (hPP) by RFamides at rabbit kidney membranes. The competition by C-terminal hexapeptide of hPP (LTRPRY.NH2) did not show such affinity change. The steady-state binding of hPP showed little sensitivity to any of the RFamides tested. The Y1-selective binding of [125I][Leu31,Pro34]hPYY (at 2 nM hPP) was much less sensitive to RFamides than the binding of [125I]hPP, albeit with some differences across tissue or cell types. The binding of Y2-selective agonist 125I-labeled human peptide YY (3-36) was quite insensitive to RFamides. The presence of a unique component in the inhibition of hPP binding by RFamides was further indicated by a degree of antagonism with phospholipase C inhibitor U-73122, and by an only limited cooperation with a N5-amiloride compound, and with alkylator chloroethylclonidine. Change of the chirality of individual residues in the FMRFamide molecule produced a significant reduction of inhibitory potency only with D-Phe in the C-terminal position. Substitution of the (C-3) L-Met by L-Leu greatly increased the inhibitory potency of RFamides relative to otherwise identical congeners. RFamides could act both as ligands of membrane neighbors of the PP receptor, and as competitors of Y5-like NPY receptor epitopes that accommodate the C-terminal aspects of agonist peptides.     

Receptors for NPY in peripheral tissues bioassays

Neuropeptide Y (NPY) and its congeners, peptide YY (PYY) and the pancreatic polypeptide (PP), have a large spectrum of peripheral actions. NPY is found in peripheral neurons, co-localized or not with noradrenaline; PYY and PP are expressed in endocrine cells of the pancreas and in the intestine of vertebrates. NPY is the most abundant peptide in the brain and is involved in the regulation of food intake and of circadian rhythm. It intervenes also in the process of anxiety and memory. NPY is a potent vasoconstrictor, a cardiac stimulant, and may affect the gut through enteric neurons. PYY and PP act mainly on the gastrointestinal system; however, when in blood, they can cross-react with functional sites elsewhere and replace NPY in some parts of the brain (e.g. regions involved in feeding behavior). These peptides act through G protein coupled receptors (GPCR) of which five different types are known and have been cloned (1,2); functional sites (receptors) for NPY have been found in vessels, the gut, and in vasa deferentia (3-6).     

Recognition of neuropeptides FMRFamide and LPLRFamide by chicken cerebellum avian pancreatic polypeptide binding sites

Binding isotherms were constructed for the binding of synthetic tetrapeptide and pentapeptide fragments to membranes prepared from chicken cerebellar tissue. Both the tetrapeptide (FMRFamide), which was originally isolated from ganglia of mollusks, and the pentapeptide (LPLRFamide) previously isolated from chicken brain are known to increase blood pressure and modulate brain neurons in rats. The C-terminal dipeptide sequences of the two peptides are identical and both show similarity to the dipeptide sequence established for the pancreatic polypeptide (PP) family. Specific high-affinity binding sites exist for the latter peptide, sites which are competed for (though with less affinity) by neuropeptide Y (NPY). Affinity for cerebellar membranes was virtually equivalent for the synthetic peptide LPLRFamide and FRMFamide; the binding affinities (IC50) of all fragments tested (C-terminal pentapeptides of avian PP and NPY, and FMRFamide and LPLRFamide) fell in the same approximate range. Since the N-terminal residues of FMRFamide and LPLRFamide are not homologous with equivalent residues of APP or NPY, our results indicate that only Arg-Tyr-NH2 or Arg-Phe-NH2 sequences are necessary for binding of the carboxy terminus peptides of the PP family. In this respect, these sequences are functionally equivalent.     

Functional CCK-A and Y2 receptors in guinea pig esophagus

Effects of cholecystokinin octapeptide (CCK-8), peptide YY (PPY), neuropeptide Y (NPY) and their analogs on muscle contractions of esophageal strips were investigated. CCK-8 induced a tetrodotoxin and atropine-sensitive contraction. The relative potencies for CCK related peptides to induce contractions were CCK-8 > desulfated CCK-8 > gastrin-17-I. The CCK-A receptor antagonist L-364,718 was 300-fold more potent than the CCK-B receptor antagonist L-365,260 at inhibiting CCK-8-induced contraction. These indicate that neural CCK-A receptors mediate this contraction. PYY or NPY did not cause muscle contraction or inhibit muscle contraction induced by carbachol, endothelin-1 or KCl. However, both PYY and NPY concentration-dependently inhibited contraction induced by CCK-8. This inhibition was not affected by nitric oxide (NO) synthase inhibitors L-NMMA or L-NAME. The relative potencies of PYY related peptides to inhibit CCK-8 induced contraction were PYY > NPY > NPY13-36 > [Leu(31), Pro(34)]NPY > pancreatic polypeptide (PP). We conclude that CCK interacts with neural CCK-A receptors to cause esophageal muscle contraction. PYY and NPY interact with Y2 receptors to inhibit this CCK-induced muscle contraction by an effect not related to NO.     

The first selective agonist for the neuropeptide YY5 receptor increases food intake in rats

The first Y(5) receptor-selective analog of neuropeptide Y (NPY), [Ala(31),Aib(32)]NPY, has been developed and biologically characterized. Using competition binding assays on cell lines that express different Y receptors, we determined the affinity of this analog to be 6 nm at the human Y(5) receptor, >500 nm at the Y(1) and Y(2) receptors, and >1000 nm at the Y(4) receptor. Activity studies performed in vitro using a cAMP enzyme immunoassay, and in vivo using food intake studies in rats, showed that the peptide acted as an agonist. Further peptides obtained by the combination of the Ala(31)-Aib(32) motif with chimeric peptides containing segments of NPY and pancreatic polypeptide displayed the same selectivity and even higher affinity (up to 0.2 nm) for the Y(5) receptor. In vivo administration of the new Y(5) receptor-selective agonists significantly stimulated feeding in rats. The NMR solution structures of NPY and [Ala(31),Aib(32)]NPY showed a different conformation in the C-terminal region, where the alpha-helix of NPY was substituted by a more flexible, 3(10)-helical turn structure.     

Pancreatic polypeptide is secreted from and controls differentiation through its specific receptors in osteoblastic MC3T3-E1 cells

Although the neuropeptide Y (NPY) family has been demonstrated to control bone metabolism, the role of pancreatic polypeptide (PP), which has structural homology with NPY and peptide YY (PYY) to share the NPY family receptors, in peripheral bone tissues has remained unknown. In the present study, we studied the regulatory roles of PP and its Y receptors using MC3T3-E1 cells, a murine transformed osteoblastic cell line, as a model for osteoblastic differentiation. We found that (1) PP mRNA was detected and increased during cell-contact-induced differentiation in MC3T3-E1 cells; (2) the immunoreactivity of PP was detected by radioimmunoassay and increased in culture medium during differentiation; (3) all the types of NPY family receptor mRNAs (Y1, Y2, Y4, Y5, and y6) were found to increase during differentiation; (4) PP stimulated differentiation in MC3T3-E1 cells in terms of ALP mRNA and BMP-2 mRNA. These findings suggested that MC3T3-E1 cells produce and secrete PP, which may in turn stimulate the differentiation of MC3T3-E1 through its specific receptors in an autocrine manner.     

Receptors on phaeochromocytoma cells for two members of the PP-fold family--NPY and PP

Pancreatic polypeptide (PP) and neuropeptide Y (NPY) belong to a family of regulatory peptides which hold a distinct tertiary structure, the PP-fold, even in dilute aqueous solution. High-affinity receptors, specific for both PP and NPY, are described on the rat phaeochromocytoma cell line, PC-12. The binding of [125I-Tyr36]PP to PC-12 cells was inhibited by concentrations of unlabeled PP which correspond to physiological concentrations of the hormone, 10(-11)-10(-9) mol/l. The affinity of the receptor for the neuropeptide, NPY, was 10(2)-times lower than that of the PP receptor. C-terminal fragments of both PP (PP24-36) and NPY (NPY13-36) were between 10(2)- and 10(3)-times less potent in displacing the radiolabeled 36-amino-acid peptides from their respective receptors. It is concluded that PC-12 cells are suited for structure-function studies of the PP-fold peptides and studies on the cellular events following cellular binding of PP-fold peptides.     

Cardiac function in neuropeptide Y Y4 receptor-knockout mice

Autonomic control of cardiovascular function in neuropeptide Y (NPY) Y4 receptor-knockout mice was investigated using pancreatic polypeptide (PP), NPY and specific agonists and antagonists for other NPY receptors well characterised in cardiovascular function. Y4 receptor-knockout mice, anaesthetised with sodium pentobarbitone, displayed slower heart rate, indicated by a higher pulse interval and lower blood pressure compared to control mice. After vagus nerves were cut heart rate increased but was still significantly slower than in control mice. PP had no effect on blood pressure or cardiac vagal activity in either group of mice, which was consistent with earlier studies in other species. Injection of NPY evoked an increase in blood pressure but the response was significantly reduced in Y4 receptor-knockout mice compared to the controls. The reduction in pressor activity was not Y1 mediated as the selective Y1 antagonist, BIBP 3226, was effective in blocking NPY pressor activity in knockout mice. In addition, cardiac vagal inhibitory activity evoked by low doses of NPY was also reduced when compared to control responses. As N-acetyl [Leu(28, 31)] NPY 24-36 inhibited vagal activity dose dependently in both groups of mice with no difference in response at any dose, it is unlikely that this effect also is receptor mediated. We propose that the reduced vasoconstrictor and vagal inhibitory activity evoked by NPY in Y4 receptor-knockout mice is due to a lack of adrenergic tone bought about by a proposed reduction in sympathetic activity, possibly resulting from altered NPY activity secondarily affecting adrenergic transmission. We conclude that Y4 receptor deletion disrupts autonomic balance within the cardiovascular system.     

Influence of neuropeptide Y and pancreatic polypeptide on islet function and beta-cell survival

BackgroundIn the present study we assessed the impact of neuropeptide Y receptor (NPYR) modulators, neuropeptide Y (NPY) and pancreatic polypeptide (PP), on islet function and beta-cell survival.MethodsThe effects of NPY and PP on beta-cell function were examined in BRIN BD11 and 1.1B4 beta-cells, as well as isolated mouse islets. Involvement of both peptides in pancreatic islet adaptations to streptozotocin and hydrocortisone, as well as effects on beta-cell proliferation and apoptosis was also evaluated.ResultsNeither NPY nor PP affected in vivo glucose disposal or insulin secretion in mice. However, both peptides inhibited (p < 0.05 to p < 0.001) glucose stimulated insulin secretion from rat and human beta-cells. NPY exerted similar insulinostatic effects in isolated mouse islets. NPY and PP inhibited alanine-induced changes in BRIN BD11 cell membrane potential and (Ca^2 +)_i. Streptozotocin treatment decreased and hydrocortisone treatment increased beta-cell mass in mice. In addition, streptozotocin, but not hydrocortisone, increased PP cell area. Streptozotocin also shifted the normal co-localisation of NPY with PP, towards more pronounced co-expression with somatostatin in delta-cells. Both streptozotocin and hydrocortisone increased pancreatic exocrine expression of NPY. More detailed in vitro investigations revealed that NPY, but not PP, augmented (p < 0.01) BRIN BD11 beta-cell proliferation. In addition, both peptides exerted protective effects against streptozotocin-induced DNA damage in beta-cells.ConclusionThese data emphasise the involvement of PP, and particularly NPY, in the regulation of beta-cell mass and function.General significanceModulation of PP and NPY signalling is suitable for further evaluation and possible clinical development for the treatment of diabetes.     

2-36[K4,RYYSA(19-23)]PP a novel Y5-receptor preferring ligand with strong stimulatory effect on food intake

Members of the neuropeptide Y (NPY) family regulate many physiological processes via interaction with at least four functional, pharmacologically distinct Y-receptors. However, selective antagonists developed for several subtypes have not been useful in defining particular Y-receptor functions in vivo. To identify critical residues within members of the NPY family required for Y-receptor subtype-selectivity we have determined the contribution of each residue within NPY to receptor binding by replacing them with L-alanine. In a second study, chimeric peptides where single or stretches of residues were interchanged between members of the NPY family were generated and tested in radioligand binding studies. Overall, substituted alanine analogues exhibited similar orders of affinities at each Y-receptor subtype with no obvious subtype-selectivity. Residues of particular interest are Leu30 which exhibited selectivity for the Y4-receptor, whereas Asp16 does not appear to play any role in ligand binding. Several chimeric peptides, e.g., [K4]pancreatic polypeptide ([K4]PP) and [RYYSA(19-23)]PP clearly showed higher affinity at the Y4 and Y5 subtypes compared to the Y1 and Y2 subtypes. In addition, the transfer of a proline residue from position 14 to 13 in peptide YY decreases its affinity at the Y1-, Y4- and Y5-receptors but is unchanged at the Y2 subtype. Combining these results, and with the help of molecular modelling, second generation chimeras were designed. The most significant improvement was achieved in chimera 2-36[K4,RYYSA(19-23)]PP where the affinity for the Y5 subtype increased by ninefold over that from NPY. Several of these compounds were also tested for their ability to stimulate food intake in a rat model. Interestingly, again 2-36[K4,RYYSA(19-23)]PP showed the most dramatic effect with a major increase on food intake over a range of doses compared to NPY suggesting a possible synergistic effect of several Y-receptors on feeding behaviour.