NEUROPEPTIDE Y AND CARDIOVASCULAR REGULATION

1. The effects of neuropeptide Y (NPY) and related peptide fragments on blood pressure and vagal action at the heart were compared in the anaesthetized rat. 2. A change in vagal action was taken as a measure of presynaptic activity and a change in blood pressure was taken as a measure of postsynaptic activity. 3. NPY, NPY-(13-36) and a stabilized 13-36 analogue of NPY (ANA NPY) all exerted pressor actions and attenuated vagal action at the heart. 4. On the basis of different potencies demonstrated for the pressor and vagal inhibitory actions of these peptides, the results are consistent with the proposal that there are two populations of NPY receptors.     

Structure-function studies on neuropeptide Y and pancreatic polypeptide--evidence for two PP-fold receptors in vas deferens

The biological effects of neuropeptide Y (NPY), rat pancreatic polypeptide (rPP), hybrid analogs of NPY and PP, and C-terminal fragments of NPY were studied in the field-stimulated rat vas deferens model. The results were correlated with peptide binding experiments in Y1 and PP receptor assays on rat PC-12 cells and Y2 receptors on porcine hippocampal membranes. NPY and rPP inhibited the electrically induced contractions in the vas deferens with an IC50 of 25 and 22 nM respectively. However, in contrast to NPY, rPP could not totally block muscle activity. The inhibitory action of the long C-terminal fragment of NPY, NPY-(19-36) and NPY-(11-36), indicated that NPY acts through a Y2 receptor in the vas deferens. The structural basis for the differential recognition of NPY and PP by Y2 receptors and partly also by PP receptors, could be defined with hybrid analogs of PP and NPY. The analogs, [Ile31,Gln34]PP and [Leu31,Pro33]NPY reacted in the vas deferens preparation in accordance with their relative potency in the Y2 and PP receptor assays. [Ile31,Gln34]PP, which bound to the Y2 receptor like NPY, was also able to block the part of the contractile response which was resistant to rPP. It is concluded that in the vas deferens, PP-fold peptides act through two types of receptors: Y2 and PP, and that residues in the C-terminal part of the molecules determine the differential recognition of the peptides by these receptor types.     

Binding properties of three neuropeptide Y receptor subtypes from zebrafish: comparison with mammalian Y1 receptors

Neuropeptide Y (NPY) and peptide YY (PYY) are two related 36-amino-acid peptides found in all vertebrates and are involved in many physiological processes. Five receptor subtypes have been cloned in mammals (Y1, Y2, Y4, Y5, and y6). We have recently cloned three NPY/PYY receptor subtypes in zebrafish, called Ya, Yb, and Yc. Here we report on a direct comparison of the pharmacological properties of these three receptors in vitro using porcine NPY with alanine substitutions in positions 33–36 as ligands and three analogues with internal deletions: [Ahx^8–20]NPY, [Ahx^8–20, Pro³⁴]NPY, and [Ahx^5–24]NPY. In all cases, the zYc receptor was the most sensitive to the modifications of the NPY molecule and zYa was the least sensitive (except for the Arg → Ala replacement at position 33). Our data identified zYa as a receptor that can bind ligands specific for Y1, Y2, and Y4 receptors, while zYb and zYc were more Y1-like. All peptides with internal deletions bound to the zYa receptor with affinities similar to that of intact pNPY. Neither the Y1-selective antagonists BIBP3226 and SR120819A nor the Y2-selective BIIE0246 bound to any of the zebrafish receptors, although the amino acids identified as important for BIBP3226 binding were almost completely conserved. These results may prove helpful in molecular modeling of the three-dimensional receptor structure.     

Structure activity and molecular modeling analyses of ribose- and base-modified uridine 5'-triphosphate analogues at the human P2Y2 and P2Y4 receptors

With the long-term goal of developing receptor subtype-selective high affinity agonists for the uracil nucleotide-activated P2Y receptors we have carried out a series of structure activity and molecular modeling studies of the human P2Y2 and P2Y4 receptors. UTP analogues with substitutions in the 2'-position of the ribose moiety retained capacity to activate both P2Y2 and P2Y4 receptors. Certain of these analogues were equieffective for activation of both receptors whereas 2'-amino-2'-deoxy-UTP exhibited higher potency for the P2Y2 receptor and 2'-azido-UTP exhibited higher potency for the P2Y4 receptor. 4-Thio substitution of the uracil base resulted in a UTP analogue with increased potency relative to UTP for activation of both the P2Y2 and P2Y4 receptors. In contrast, 2-thio substitution and halo- or alkyl substitution in the 5-position of the uracil base resulted in molecules that were 3-30-fold more potent at the P2Y2 receptor than P2Y4 receptor. 6-Aza-UTP was a P2Y2 receptor agonist that exhibited no activity at the P2Y4 receptor. Stereoisomers of UTPalphaS and 2'-deoxy-UTPalphaS were more potent at the P2Y2 than P2Y4 receptor, and the R-configuration was favored at both receptors. Molecular docking studies revealed that the binding mode of UTP is similar for both the P2Y2 and P2Y4 receptor binding pockets with the most prominent dissimilarities of the two receptors located in the second transmembrane domain (V90 in the P2Y2 receptor and I92 in the P2Y4 receptor) and the second extracellular loop (T182 in the P2Y2 receptor and L184 in the P2Y4 receptor). In summary, this work reveals substitutions in UTP that differentially affect agonist activity at P2Y2 versus P2Y4 receptors and in combination with molecular modeling studies should lead to chemical synthesis of new receptor subtype-selective drugs.     

Enhanced neuropeptide Y immunoreactivity and vasoconstriction in mesenteric small arteries from the early non-obese diabetic mouse

The present study investigated whether sympathetic neurotransmission is altered at an early stage of diabetes in mesenteric small arteries isolated from female non-obese diabetic (NOD) and control animals without diabetes from the same mouse strain. The NOD diabetic mice had increased plasma glucose and hypertension. Confocal microscopy showed distribution of nerve terminals was similar, but immunoreaction intensity for neuropeptide Y (NPY) and tyrosine hydroxylase was higher in small arteries from NOD diabetic compared with NOD control mice. In the presence of prazosin and activated with vasopressin, electrical field stimulation evoked contractions which were more pronounced in mesenteric arteries from NOD diabetic versus NOD control mice and inhibited by the NPY Y(1) receptor antagonist, BIBP 3226. NPY concentration-response curves were leftward shifted in arteries from NOD diabetic versus NOD control both in arteries with and without endothelium, but not in the presence of the BIBP 3226. The present findings suggest that enhanced NPY content and vasoconstriction to NPY by activation of NPY Y(1) receptors in arteries from diabetic mice may contribute to the enhanced sympathetic nerve activity and vascular resistance in female non-obese early diabetic animals.     

Co-expression of neuropeptide Y Y1 and Y5 receptors results in heterodimerization and altered functional properties

Centrally administered neuropeptide Y (NPY) produces anxiolytic and orexigenic effects by interacting with Y1 and Y5 receptors that are colocalized in many brain regions. Therefore, we tested the hypothesis that co-expression of Y1 and Y5 receptors results in heterodimerization, altered pharmacological properties and altered desensitization. To accomplish this, the carboxyl-termini of Y1 and Y5 receptors were fused with Renilla luciferase and green fluorescent protein and the proximity of the tagged receptors assessed using bioluminescent resonance energy transfer. Under basal conditions, cotransfection of tagged Y1 receptor and Y5 produced a substantial dimerization signal that was unaffected by the endogenous, nonselective agonists, NPY and peptide YY (PYY). Selective Y5 agonists produced an increase in the dimerization signal while Y5 antagonists also produced a slight but significant increase. In the absence of agonists, selective antagonists decreased dimerization. In functional studies, Y5 agonists produced a greater inhibition of adenylyl cyclase activity in Y1/Y5 cells than cells expressing Y5 alone while NPY and PYY exhibited no difference. With PYY stimulation, the Y1 antagonist became inactive and the Y5 antagonist exhibited uncompetitive kinetics in the Y1/Y5 cell line. In confocal microscopy studies, Y1/Y5 co-expression resulted in increased Y5 signaling following PYY stimulation. Addition of both Y1 and Y5 receptor antagonists was required to significantly decrease PYY-induced internalization. Therefore, Y1/Y5 co-expression results in heterodimerization, altered agonist and antagonist responses and reduced internalization rate. These results may account for the complex pharmacology observed when assessing the responses to NPY and analogs in vivo.     

Neuropeptide Y1 subtype pharmacology of a recombinantly expressed neuropeptide receptor.

Neuropeptide Y (NPY) is an important central and peripheral modulator of neural and endocrine functions. This neuropeptide interacts with at least two pharmacologically distinct receptors, termed Y1 and Y2. At Y1 receptors, the NPY analog [Leu31,Pro34] NPY, but not the carboxyl-terminal fragment NPY-(18-36), displaces radiolabeled NPY and the sequence-related peptide YY, whereas Y2 receptors exhibit the opposite selectivity. We have used cultured mammalian 293 cells for the high level transient expression of a previously cloned putative neuropeptide receptor of rat brain. We report that this receptor displays the ligand binding properties and selectivity of a Y1 receptor, with a single high affinity site for 125I-NPY (Kd, 0.7 +/- 0.2 nM). The functionality of the recombinantly expressed receptor was demonstrated by an inhibition of adenylyl cyclase and a concomitant mobilization of intracellular Ca2+.     

Neuropeptide Y receptor subtypes in the basolateral nucleus of the amygdala modulate anxiogenic responses in rats

The behavioral effects induced by intra-amygdala stimulation of the neuropeptide Y (NPY) Y(2) and the NPY Y(5) receptor subtypes were assessed in the social interaction (SI) test. Microinjections of NPY(3-36), an NPY Y(2) preferring agonist, into the basolateral nucleus of the amygdala (BLA) produced bi-directional dose-response curve. At low doses NPY(3-36) has an anxiogenic effect while at higher doses it produced an anxiolytic effect. Pretreatment with the NPY Y(5) receptor antagonist Novartis 1(1 nmol), an analog of CGP71683A synthesized by Eli Lilly and Company, IN, blocked the anxiolytic effects of NPY(3-36) (80 pmol), while pretreatment with BIBO 3304 (200 pmol), a Y(1) antagonist, had no effect, suggesting that the Y(5), but not the Y(1) receptor was involved in the anxiolytic behavior produced following intra-amygdalar NPY(3-36) administration. In addition, the Y(5) antagonist had no behavioral effect when given alone at 1.0 nmol. These findings support the hypothesis that amygdalar Y(2) receptors may play a role in mediating anxiogenic effects, while Y(5) receptors may be involved in the anxiolytic behaviors of NPY.     

Neuropeptide Y inhibits potassium-stimulated glutamate release through Y2 receptors in rat hippocampal slices in vitro.

1. We investigated the effects of neuropeptide Y (NPY), peptide YY (PYY), NPY13-36, NPY18-36, [Leu31][Pro34]NPY and of pancreatic polypeptide Y (PPY) on calcium-dependent, potassium-stimulated glutamate release in superfused rat hippocampal slices. 2. NPY, PYY and the Y2 receptor agonist NPY13-36 equipotently inhibited the release of glutamate. The half-maximal response was observed at about 10 nM in a dose-dependent manner (3 to 100 nM). Maximal inhibition of 50 to 60% was obtained at 100 nM. At higher concentrations of the peptides (300 nM and 1 microM) this inhibition was partially or entirely reversed. Porcine NPY13-36 and NPY18-36 inhibited glutamate release by about 44% at 100 nM. 3. The specific Y1 receptor agonist, [Leu31][Pro34]NPY, caused an insignificant increase in glutamate release at 100 to 300 nM concentrations. PPY had no effect on potassium-evoked glutamate release in hippocampal slices at concentrations of 30 nM to 1 microM. 4. The experiments support previous electrophysiological data. They suggest a potent inhibitory action of NPY through NPY-Y2 receptors on the release of the excitatory amino acid glutamate in rat hippocampus. Especially under conditions of increased NPY synthesis, such as in epilepsy, this mechanism may be of pathophysiological relevance.     

INHIBITORY EFFECT OF NEUROPEPTIDE Y ON STIMULATED RENIN SECRETION OF AWAKE RATS

1. (1-36)-NPY is a vasoconstrictor peptide widely distributed in sympathetic nerve terminals. This peptide exerts an inhibitory action on renin release induced by various stimuli. Post-synaptic neuropeptide Y (NPY) receptors show a high affinity for (1-36)-NPY as well as for the agonist (Pro34)-NPY, while presynaptic receptors bind preferentially (13-36)-NPY. 2. This study was undertaken to assess whether the NPY induced renin suppression in awake normotensive rats infused with the beta-adrenoceptor stimulant isoproterenol is mediated by activation of pre- or post-synaptic receptors. 3. Non-pressor doses of (1-36)-NPY and (Pro34)-NPY markedly attenuated the renin secretion triggered by isoproterenol whereas (13-36)-NPY had no effect. This suggests that the effect of NPY on renin release is due to the stimulation of post-synaptic receptors. However it remains unknown whether NPY acts directly on juxtaglomerular cells or indirectly by modifying intraglomerular haemodynamics.     

Neuropeptide Y: identification of the binding site

Based on the hypothetical 3D structure of neuropeptide Y (NPY), NPY 1-4-Aca-25-36, a 17 amino acid analogue, has been synthesized replacing the sequence NPY 5-24 by ε-aminocaproic acid (Aca). This low-molecular weight deletion analogue showed nearly comparable receptor affinity to NPY. In order to elucidate the structural requirements for receptor recognition each amino acid of 1-4-Aca-25-36 was exchanged by its D-enantiomer, glycine and L-alanine. In addition distinct amino acids were replaced by closely related residues. Multiple peptide synthesis was applied using Fmoc-strategy and BOP activation. Binding assay was performed on rabbit kidney membrane preparations. The results of structure affinity studies suggest that the C-terminal tetrapeptide NPY 33-36 is essential for receptor recognition.     

Design and Synthesis of Peptide YY Analogues with C-terminal Backbone Amide-to-Ester Modifications

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Different neuropeptide Y receptor subtypes in rat and rabbit vas deferens.

Prejunctional neuropeptide Y (NPY) receptors that inhibit the contractions evoked in rat and rabbit vas deferens by field stimulation were investigated by using NPY, [Leu31,Pro34]NPY and the fragments, NPY-(13-36) and NPY-(18-36). NPY, and especially [Leu31,Pro34]NPY, were more potent agonists on the twitch response of the rabbit vas deferens. In contrast the NPY C-terminal fragments, NPY-(13-36) and NPY-(18-36), inhibited the twitch response at lower concentrations in the rat vas deferens. These results indicate that distinct NPY receptor subtypes mediate the biological effect in these two tissues. We suggest that prejunctional receptors in the rat vas deferens are of the Y2-subtype and those in rabbit vas deferens of the Y1-subtype.     

Presynaptic inhibition by neuropeptide Y in rat hippocampal slice in vitro is mediated by a Y2 receptor.

1. The action of analogues and C-terminal fragments of neuropeptide Y (NPY) was examined on excitatory synaptic transmission in area CA1 of the rat hippocampal slice in vitro, by use of intracellular and extracellular recordings, to determine by agonist profile the NPY receptor subtype mediating presynaptic inhibition. 2. Neither NPY, analogues nor fragments of NPY affected the passive or active properties of the post-synaptic CA1 pyramidal neurones, indicating their action is at a presynaptic site. 3. The full-sequence analogues, peptide YY (PYY) and human NPY (hNPY), were equipotent with NPY at the presynaptic receptor, while desamido hNPY was without activity. 4. NPY2-36 was equipotent with NPY. Fragments as short as NPY 13-36 were active, but gradually lost activity with decreasing length. NPY 16-36 had no effect on extracellular field potentials, but still significantly inhibited excitatory postsynaptic potential amplitudes. Fragments shorter than NPY 16-36 had no measurable effect on synaptic transmission. 5. The presynaptic NPY receptor in hippocampal CA1 therefore shares an identical agonist profile with the presynaptic Y2 receptor at the peripheral sympathetic neuroeffector junction.     

Evidence for two neuropeptide Y receptors mediating vasoconstriction.

The presence of receptor subtypes mediating the vascular and prejunctional effects of neuropeptide Y (NPY) was investigated using the Y2 receptor agonist, NPY-(13-36), and the Y1 agonist, [Leu31,Pro34]NPY. NPY-(1-36) and [Leu31,Pro34]NPY administered i.v. to anesthetized pigs evoked dose-dependent increases in mean arterial blood pressure and splenic and renal vascular resistance, and a decrease in heart rate. The potency of [Leu31,Pro34]NPY was 10-30% that of NPY-(1-36). In the spleen, NPY-(13-36) evoked vasoconstriction similar to that evoked by [Leu31,Pro34]NPY, but did not significantly increase renal vascular resistance or mean arterial blood pressure. Local intra-arterial administration of [Leu31,Pro34]NPY caused an increase in nasal mucosal vascular resistance with a potency similar to that of NPY-(13-36) evoked only a minor (17%) increase in nasal mucosal vascular resistance. The NPY analogues were further characterized in receptor binding studies on pig spleen membranes. Compared to NPY-(1-36), 800 times higher concentrations of [Leu31,Pro34]NPY, and 7 times higher concentrations of NPY-(13-36) were required to achieve the same 50% displacement of [125I]NPY-(1-36). Electrically evoked contractions in rat vas deferens were inhibited by 50% by 0.05 microM NPY-(1-36) and 0.3 microM NPY-(13-36), while [Leu31,Pro34]NPY only slightly attenuated the contractions (by 24% at 1 microM). The present data suggest the existence of subtypes of NPY receptors mediating vasoconstriction. Thus, the splenic vascular bed of the pig contains both Y1 and Y2 receptors while the Y1 subtype predominates in the kidney, nasal mucosa and for blood pressure control. The prejunctional receptor in rat vas deferens seems to be of the Y2 subtype.     

Evidence for involvement of neuropeptide Y receptors in the regulation of food intake: studies with Y1-selective antagonist BIBP3226

1. Experiments were conducted to evaluate the effects of the novel non-peptide neuropeptide Y Y₁ receptor antagonist, BIBP3226 (N²-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide) on spontaneous, fasting-induced and NPY-induced food intake in rats. In addition to consumption of regular chow, the effects of BIBP3226 on consumption of highly palatable sweetened mash were monitored in a 1 h test on first exposure and after familiarization with novel food.
2. BIBP3226 (10.0 nmol, i.c.v.) had no effect on the consumption of regular chow, but reduced significantly the intake of highly palatable diet and the food intake stimulated by fasting (24 h). Neuropeptide Y (NPY, 1.0 nmol, i.c.v.) significantly increased the consumption of regular rat chow. This orexigenic effect of NPY was blocked by BIBP3226 (10.0 nmol, administered i.c.v. 5 min before NPY) at 30  min and 4  h, but not at 1 and 2  h. When animals were pretreated with diazepam (0.5 mg kg⁻¹, i.p., 20 min before NPY), BIBP3226 failed to suppress NPY-induced feeding.
3. An NPY Y₁ and Y₃ receptor agonist, [Leu³¹,Pro³⁴]NPY and a Y₅ receptor agonist human peptide YY_3–36 (hPYY_3–36, both 30 pmol), microinjected into the paraventricular nucleus of the hypothalamus (PVN) increased the consumption of regular rat chow. BIBP3226 (0.4 nmol, into the PVN) completely blocked the stimulatory effect of [Leu³¹,Pro³⁴]NPY but not that of hPYY_3–36. BIBP3226 (0.4 nmol) alone failed to modify the consumption of the regular chow. Higher doses of BIBP3226 (1.0 and 2.0 nmol) injected into the vicinity of the PVN reduced the consumption of the sweetened mash.
4. These results suggest that both the NPY Y₁ and Y₅ receptors in the PVN are involved in the regulation of food intake. The stimulatory effect of exogenous NPY is probably mediated through an NPY receptor subtype that is not identical with the Y₁ receptor (possibly Y₅ receptor). However, the NPY Y₁ receptors may mediate the effect of endogenous NPY in conditions of increased energy demand or on intake of highly palatable diets.

     

Neuropeptide Y inhibits Ca2+ influx into cultured dorsal root ganglion neurones of the rat via a Y2 receptor.

1. The identity of the neuropeptide Y (NPY) receptor associated with the observed inhibition of neuronal Ca2+ currents (ICa) in rat dorsal root ganglion (DRG) cells has been established on the basis of agonist responses to analogues and carboxy terminal (C-terminal) fragments of the NPY molecule. 2. Whole cell barium currents (IBa) in DRG cells were reversibly inhibited by 100 nM NPY, 100 nM PYY and C-terminal fragments of NPY in a manner that correlated with the length of the NPY fragments (for inhibition of the IBa NPY = PYY greater than NPY2-36 greater than NPY13-36 greater than NPY16-36 greater than NPY18-36 much greater than NPY25-36). 3. C-terminal fragments of NPY were also effective in reversibly reducing the ICa, the associated increase in the intracellular Ca2+ concentration [( Ca2+]i) and the increased [Ca2+]i produced by evoked action potentials in the DRG cells. In addition, a Ca(2+)-activated Cl- conductance was also reversibly reduced by NPY fragments only when accompanied by a reduction in Ca2+ entry. 4. We conclude that the Y2 receptor for neuropeptide Y is coupled to inhibition of Ca2+ influx via voltage-sensitive calcium channels in DRG cells.     

Synthesis of porcine neuropeptide Y(NPY) in solution

The porcine neuropeptide Y (NPY), a 36-residue peptide amide, was synthesized by assembling six peptide fragments followed by thioanisole-mediated deprotection with trifluoromethanesulfonic acid in trifluoroacetic acid. β-Cycloheptyl aspartate, Asp(OChp), was employed to suppress base-catalyzed succinimide formation. When administered to dogs, the purified peptide (10μg/kg) caused prolonged increase of systemic arterial blood pressure and decreased pancreatic blood flow.     

The role of NPY in metabolic homeostasis: implications for obesity therapy

Neuropeptide Y (NPY) is a 36 amino acid amidated peptide which has now emerged as an important regulator of feeding behaviour. Upon intracerebroventricular (icv.) administration, NPY produces a pronounced feeding response in a variety of species. The actions of NPY are believed to be mediated by a family of receptor subtypes named Y1 - y6. Recent studies suggest that the Y1 and Y5 receptor subtypes are intimately involved in NPY induced feeding. This review presents preclinical data obtained with receptor subtype selective agonists and antagonists as well as findings from knockout mice. These new data suggest that NPY receptor antagonists may become an additional option for treating human obesity.