Central administration of neuropeptide Y induces wakefulness in rats

Neuropeptide Y (NPY) is a well-characterized neuromodulator in the central nervous system, primarily implicated in the regulation of feeding. NPY, orexins, and ghrelin form a hypothalamic food intake regulatory circuit. Orexin and ghrelin are also implicated in sleep-wake regulation. In the present experiments, we studied the sleep-modulating effects of central administration of NPY in rats. Rats received intracerebroventricular injection of physiological saline or three different doses of NPY (0.4, 2, and 10 microg in a volume of 4 microl) at light onset. Another group of rats received bilateral microinjection of saline or 2 microg NPY in the lateral hypothalamus in a volume of 0.2 microl. Sleep-wake activity and motor activity were recorded for 23 h. Food intake after the control and treatment injections was also measured on separate days. Intracerebroventricular and lateral hypothalamic administration of NPY suppressed non-rapid-eye-movement sleep and rapid-eye-movement sleep in rats during the first hour after the injection and also induced changes in electroencephalogram delta power spectra. NPY stimulated food intake in the first hour after both routes of administration. Data are consistent with the hypothesis that NPY has a role in the integration of feeding, metabolism, and sleep regulation.     

Neuropeptide Y innervation of amygdaloid and hypothalamic neurons that project to the dorsal vagal complex in rat

The anatomic relationship between neuropeptide Y (NPY)-immunoreactive terminals and forebrain areas in the rat that contain neurons that project to the dorsal vagal complex (DVC) was examined. To accomplish this, the combined retrograde fluorescent tracer and immunofluorescent technique was used. Neurons projecting to the DVC within the parvocellular divisions of the paraventricular nucleus of the hypothalamus were the most heavily innervated of the regions studied. A relatively high density of NPY-immunoreactive terminals innervated regions of the arcuate, dorsomedial and lateral hypothalamic areas that contained DVC efferent cells. Neurons that projected to the DVC within the medial division of the central nucleus of the amygdala and the lateral part of the bed nucleus of the stria terminalis were also innervated by NPY immunoreactive terminals. The results suggest an important role for NPY terminals in the modulation of neurons within the amygdala and hypothalamus that directly influence visceral-autonomic functions of the dorsal vagal complex. The source and possible function of NPY within these regions is discussed.     

Internalization studies of chimeric neuropeptide Y receptors Y1 and Y2 suggest complex interactions between cytoplasmic domains

Agonist stimulation readily internalizes neuropeptide Y receptor Y1 while there are contradictory results for the Y2 receptor. In order to explore putative functional differences between the Y1 and Y2 receptors we generated reciprocal chimeras by swapping the third intracellular loop, the carboxy terminus or both between human Y1 and Y2. Internalization was studied in a quantitative radioligand binding assay with removal of surface-bound ligand in an acidic-wash procedure. The internalization assay revealed a lower degree of internalization as well as slower kinetics for the Y2 receptor. Generally, reciprocal exchange of receptor segments did not convey properties of the donor receptor but tended to enhance internalization. Surprisingly, insertion of the Y2 carboxy terminus into Y1 gave almost complete internalization (92%), rather than reduced internalization, while the insertion of both segments resulted in internalization equal to the native Y1 receptor. These findings were confirmed by fluorescence microscopy of immuno-stained receptors tagged with a C-terminal FLAG epitope. However, after exposure to high agonist concentrations (100 nM) Y2 was internalized. Studies of Y2 and the closely related Y7 receptor confirmed low internalization for Y2 from chicken and teleost fishes as well as Y7 from two teleosts. The conservation across species of low internalization at physiological concentrations suggests that this is an ancient feature and of vital importance for Y2 function. We propose that amino acid motifs in the third intracellular loop as well as the C terminus of both Y1 and Y2 are able to drive agonist-promoted internalization and that there may be constraining motifs in the Y2 receptor.     

Centrally administered neuropeptide Y delays gastric emptying via Y2 receptors in rats

It has been shown that centrally administered neuropeptide Y (NPY) delays gastric emptying. To determine the receptor subtypes of NPY mediating the inhibitory effects on gastric emptying, effects of intracerebroventricular injection of NPY, [Leu31,Pro34]NPY (a Y1 agonist) and NPY-(3-36) (a Y2 agonist) on solid gastric emptying and postprandial antropyloric motility were studied in conscious rats. Intracerebroventricular injection of NPY and NPY-(3-36), but not [Leu31,Pro34] NPY, delayed solid gastric emptying in a dose-dependent manner (0.03-3 nmol). After the feeding (40 min), contractions with low frequency and high amplitude of the antrum were frequently observed, and the peak contraction of the antrum occurred most often 3-6 s before the peak contraction of the pylorus. Intracerebroventricular injection of NPY and NPY-(3-36) (3 nmol), but not [Leu31,Pro34]NPY, significantly reduced antral contractions and the number of antropyloric coordination events. It is suggested that centrally administered NPY impairs postprandial antral contractions and antropyloric coordination via Y2 receptors, resulting in delayed gastric emptying.     

Distribution of neuropeptide Y Y1 and Y2 receptors in the postmortem human heart

In the present study, we present for the first time the presence and distribution of neuropeptide Y (NPY) receptors Y1 and Y2 in the human postmortem heart using specific antibodies raised against extracellular parts of the receptors. A more intensive staining against the Y2 than against the Y1 receptors was detected on both atrial and ventricular cardiomyocytes. Immunoreactivity against both receptors was identified on both conducting fibers and cardiac nerves. More vessels stained positively for the Y2 than for the Y1 receptor, but the Y1 receptors were more abundant in subendocardial than subepicardial vessels of the left ventricular wall.     

Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5

The neuropeptide Y (NPY) receptor subtypes Y1 and Y5 are involved in the regulation of feeding and several other physiological functions in mammals. To increase our understanding of the origin and mechanisms of the complex NPY system, we report here the cloning and pharmacological characterization of receptors Y1 and Y5 in the first non-mammal, chicken (Gallus gallus). The receptors display 80-83% and 64-72% amino acid sequence identity, respectively, with their mammalian orthologues. The three endogenous ligands NPY, peptide YY (PYY) and pancreatic polypeptide (PP) have similar affinities as in mammals, i.e. NPY and PYY have subnanomolar affinity for both receptors whereas chicken PP bound with nanomolar affinity to Y5 but not to Y1. A notable difference to mammalian receptor subtypes is that the Y1 antagonist SR120819A does not bind chicken Y1, whereas BIBP3226 does. The Y5 antagonist CGP71863A binds to the chicken Y5 receptor. Anatomically, both Y1 and Y5 have high mRNA expression levels in the infundibular nucleus which is the homologous structure of the hypothalamic arcuate nucleus in mammals. These results suggest that some of the selective Y1 and Y5 antagonists developed in mammals can be used to study appetite regulation in chicken.     

Cholinergic stimulation of neuropeptide Y secretion from the isolated perfused rat stomach.

Neuropeptide Y (NPY) is present in both extrinsic sympathetic adrenergic nerve terminals and intrinsic nerves of the gastrointestinal (GI) tract. Based on this localization a number of functions have been attributed to GI NPY including regulation of blood flow, intestinal fluid and electrolyte transport, and motility. There is nothing currently known, however, about the regulation of its secretion from GI nerves. The effect of cholinergic agonists and antagonists on secretion of NPY immunoreactivity (NPY-IR) from the isolated perfused rat stomach was investigated in the present study. Perfusate samples were extracted and concentrated on SepPak cartridges. Basal levels of NPY-IR varied between 98 and 147 fmol/min. Release was stimulated by high potassium concentrations (50 mM) and acetylcholine (ACh; 1 microM). ACh-induced secretion was unaffected by atropine, but inhibited by hexamethonium. Further evidence for a nicotinic component in the regulation of NPY-IR secretion was obtained by the finding of hexamethonium-induced reduction in basal secretion and stimulation of secretion by 1,1-dimethyl-4-phenyl-piperazinium (DMPP). In conclusion, cholinergic agonists and antagonists can modulate gastric NPY-IR secretion, and the cholinergic stimulatory effects are probably mediated via nicotinic receptor stimulation at the level of the intrinsic ganglia.     

Modeling of neuropeptide receptors Y1, Y4, Y5, and docking studies with neuropeptide antagonist

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.     

Differential organization of excitatory and inhibitory synapses within the rat dorsal vagal complex

The dorsal motor nucleus of the vagus (DMV) is pivotal in the regulation of upper gastrointestinal functions, including motility and both gastric and pancreatic secretion. DMV neurons receive robust GABA- and glutamatergic inputs. Microinjection of the GABA(A) antagonist bicuculline (BIC) into the DMV increases pancreatic secretion and gastric motility, whereas the glutamatergic antagonist kynurenic acid (KYN) is ineffective unless preceded by microinjection of BIC. We used whole cell patch-clamp recordings with the aim of unveiling the brain stem neurocircuitry that uses tonic GABA- and glutamatergic synapses to control the activity of DMV neurons in a brain stem slice preparation. Perfusion with BIC altered the firing frequency of 71% of DMV neurons, increasing firing frequency in 80% of the responsive neurons and decreasing firing frequency in 20%. Addition of KYN to the perfusate either decreased (52%) or increased (25%) the firing frequency of BIC-sensitive neurons. When KYN was applied first, the firing rate was decreased in 43% and increased in 21% of the neurons; further perfusion with BIC had no additional effect in the majority of neurons. Our results indicate that there are several permutations in the arrangements of GABA- and glutamatergic inputs controlling the activity of DMV neurons. Our data support the concept of brain stem neuronal circuitry that may be wired in a finely tuned organ- or function-specific manner that permits precise and discrete modulation of the vagal motor output to the gastrointestinal tract.     

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.     

Cyclooxygenase inhibition in the dorsal vagal complex of the rat evokes increases in gastric motor function

To characterize the involvement of brainstem cyclooxygenase (COX) in the vagal control of gastric motor function, tolmetin, a reversible COX inhibitor, was applied to the surface of the dorsal medulla oblongata or microinjected into the dorsal vagal complex (DVC) in β-chloralose anesthetized rats, while intragastric pressure and contractile activity of the pyloric circular and greater curvature longitudinal muscle were monitored. Tolmetin, applied to the surface of the medulla oblongata, increased intragastric pressure and stimulated contractile activity of gastric smooth muscle. Comparable gastric motor effects were observed after microinjection of tolmetin into the DVC. All the effects of tolmetin were abolished by bilateral vagotomy at the midcervical level. These results demonstrate for the first time that COX inhibition evokes vagally-mediated gastric motor effects in the DVC of the rat and support a role for COX products in gastrointestinal regulation.     

Possible involvement of neuropeptide Y Y1 receptors in antidepressant like effect of agmatine in rats

Agmatine and neuropeptide Y (NPY) are widely distributed in central nervous system and critically involved in modulation of depressive behavior in experimental animals. However their mutual interaction, if any, in regulation of depression remain largely unexplored. In the present study we explored the possible interaction between agmatine and neuropeptide Y in regulation of depression like behavior in forced swim test. We found that acute intracerebroventricular (i.c.v.) administration of agmatine (20–40 μg/rat), NPY (5 and 10 μg/rat) and NPY Y1 receptor agonist, [Leu³¹, Pro³⁴]-NPY (0.4 and 0.8 ng/rat) dose dependently decreased immobility time in forced swim test indicating their antidepressant like effects. In combination studies, the antidepressant like effect of agmatine (10 μg/rat) was significantly potentiated by NPY (1 and 5 μg/rat, icv) or [Leu³¹, Pro³⁴]-NPY (0.2 and 0.4 ng/rat, icv) pretreatment. Conversely, pretreatment of animals with NPY Y1 receptor antagonist, BIBP3226 (0.1 ng/rat, i.c.v.) completely blocked the antidepressant like effect of agmatine (20–40 μg/rat) and its synergistic effect with NPY (1 μg/rat, icv) or [Leu³¹, Pro³⁴]-NPY (0.2 ng/rat, icv). The results of the present study showed that, agmatine exerts antidepressant like effects via NPYergic system possibly mediated by the NPY Y1 receptor subtypes and suggest that interaction between agmatine and neuropeptide Y may be relevant to generate the therapeutic strategies for the treatment of depression.     

Bombesin microinjected into the dorsal vagal complex inhibits vagally stimulated gastric acid secretion in the rat

Medullary sites of action for bombesin-induced inhibition of gastric acid secretion were investigated in urethane-anesthetized rats with gastric fistula. Unilateral microinjection of bombesin or vehicle into the dorsal vagal complex was performed using a glass micropipet and pressure ejection of 100 nl volume; gastric acid output was measured every 10 min by flushing the stomach. Microinjection of vehicle into the dorsal vagal complex did not alter gastric acid secretion (1.9 +/- mumol/10) from preinjection levels (2.9 +/- 0.8 mumol/10 min). Microinjection of the stable thyrotropin-releasing hormone (TRH) analog, RX 77368, at a 77 pmol dose into the dorsal vagal complex stimulated gastric acid secretion for 100 min with a peak response at 40 min (24.1 +/- 3.2 mumol/10 min). Concomitant microinjection of RX 77368 (77 pmol) with bombesin (0.6-6.2 pmol) into the dorsal vagal complex dose dependently inhibited by 35-86% the gastric acid response to the TRH analog. Bombesin (6.2 pmol) microinjected into the dorsal vagal complex inhibited by 17% pentagastrin infusion-induced stimulation of gastric acid secretion (13.2 +/- 0.8 mumol/10 min) whereas intracisternal injection induced a 69% inhibition of the pentagastrin response. These results demonstrate that the dorsal motor complex is a sensitive site of action for bombesin-induced inhibition of vagally stimulated gastric secretion. However, other medullary sites must be involved in mediating the inhibitory effect of intracisternal bombesin on pentagastrin-stimulated gastric acid secretion.     

Neuropeptide Y stimulates DNA synthesis in human vascular smooth muscle cells through neuropeptide Y Y1 receptors

We investigated the mitogenic effect, measured as [3H]thymidine incorporation, of neuropeptide Y (NPY) on smooth muscle cells (SMCs) from human subcutaneous arteries (diameter: 0.4 mm). NPY stimulated DNA synthesis in a concentration-dependent manner, Emax 32 +/- 5% relative to control. The effect was potently antagonised by the NPY Y1 receptor antagonist BIBP3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine-a mide), indicating the effect to be mediated via the NPY Y1 receptor. Noradrenaline (NA) also induced mitogenesis, Emax 35 +/- 10% relative to control. When added together, NPY and NA potentiated the [3H]thymidine incorporation, Emax 109 +/- 38% relative to control. Also, this effect seems to be mediated by the NPY Y1 receptor, since BIBP3226 blocked the effect (44 +/- 9% relative to control). The mitogenic effect of NPY and NA, two important transmitters of the sympathetic nervous system, might have clinical consequences on conditions with elevated sympathetic nerve activity.     

Cellular injury induces activation of MAPK via P2Y receptors

Wound healing is a complex process that involves cell communication, migration, proliferation, and changes in gene expression. One of the first events after injury is the rapid release of Ca(2+) that propagates as a wave to neighboring cells (Klepeis et al. [2001]: J. Cell. Sci. 114:4185-4195). Our goal was to examine the signaling events induced by cellular injury and identify extracellular molecules that induce the activation of extracellular signal responsive kinase (ERK) (p42/44). In this study we demonstrated that injury induced ERK1/2 activation occurred within 2 min and was negligible by 15 min. Treatment of unwounded cells with wound media caused activation of ERK that could be inhibited by apyrase III. Stimulation with epidermal growth factor (EGF) did not mimic the injury response and it was not detected in the wound media. To identify the active component, size fractionation was performed and factor(s) less than 3 kDa that induced the release of Ca(2+) and activation of ERK1/2 were identified. Activity was not altered by heat denaturation, incubation with proteinase K but it was lost by treatment with apyrase. Adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), and uridine diphosphate (UDP) promoted activation by 2 min with similar profiles as that generated by injury. Preincubation with phospholipase C inhibitor, U73122, inhibited activation that was induced by injury and/or nucleotides. Lack of activation by alpha-beta-methylATP (alpha, beta-MeATP) and beta-gamma-methylATP (beta, gamma-MeATP) to purinergic (P)2X receptors further indicated that activation occurs via P2Y and not P2X purinergic receptors. These results indicate that injury-induced activation of ERK1/2 is mediated by a P2Y signaling pathway.     

Central administration of neuropeptide Y induces hypothermia in mice. Possible interaction with central noradrenergic systems

Neuropeptide Y (0.24 and 1.17 nmol icv) and clonidine (0.025, 0.05 and 0.1 mg/Kg ip) induced a slight decrease of short duration of the rectal temperature in mice in a dose-dependent manner. While pretreatment with yohimbine (0.5 mg/Kg sc), was without effect on neuropeptide Y-induced hypothermia, it attenuated the hypothermic effect of clonidine. The association of neuropeptide Y (0.05 and 0.24 nmol icv) with clonidine (0.0125, 0.025, 0.05 and 0.1 mg/Kg ip) induced a synergistic effect, but it only was significant when neuropeptide Y 0.05 and 0.24 nmol icv was associated with clonidine 0.1 mg/Kg ip and when neuropeptide Y 0.05 nmol icv was associated with clonidine 0.05 mg/Kg ip. These results suggest that the effect of neuropeptide Y is not mediated by an interaction on alpha 2-adrenoceptor, but in accordance with these results, the existence of a collaborative mechanism between both neuropeptide Yergic and noradrenergic systems cannot be ruled out.     

Distinct roles of the Y1 and Y2 receptors on neuropeptide Y-induced sensitization to sedation

Intracranial injection of neuropeptide Y (NPY) increases the sensitivity to sodium pentobarbital and ketamin sedation and has similar properties as GABA agonists on sleep. Mice sensitive to sedation have increased levels of NPY in many brain regions and Y1(-/-) mice show a marked resistance to barbiturates. Here we characterized the role of the NPY Y receptors in anesthetic-induced sedation. We show that Y1 and Y2, but not Y5, receptors participate in the modulation of sedation. Administration of a Y1 agonist increased the sodium pentobarbital-induced sedation and Y1(-/-) mice were less sensitive to this anesthetic. However, Y2(-/-) mice display increased sensitivity, showing that Y2 modulates GABAergic induced sedation both pharmacologically and physiologically and has a functionally opposing role to the Y1 receptor. Analysis of Y1(-/-)/Y2(-/-) double mutant mice show that increased sensitivity by Y1 occurs independent of the Y2 receptor, while the decreased sensitivity mediated by Y2 depend on an intact Y1 receptor. In contrast to sodium pentobarbital, both Y1 and Y2 receptors increase the sensitivity in a collaborative fashion to NMDA antagonist-induced sedation. These data demonstrate the physiological and pharmacological impact of the Y1 and Y2 receptors on sedation.