Discrimination between neuropeptide Y and peptide YY in the rat tail artery by the neuropeptide Y1-selective antagonist, BIBP 3226.

1. The ability of the novel, nonpeptide, neuropeptide Y (NPY) Y1-selective antagonist, BIBP 3226 ¿(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginine amide¿, to antagonize the increase in perfusion pressure induced by NPY and peptide Y (PYY) was tested in the perfused rat tail artery, a postjunctional Y1-receptor bioassay, precontracted by 1 microM phenylephrine. 2. NPY and PYY produced a concentration-dependent enhancement of the vasoconstrictor response evoked by 1 microM phenylephrine. Although NPY and PYY are roughly equipotent, the maximal contractile response elicited by PYY was about twice that elicited by NPY. 3. Increasing concentrations of BIBP 3226 caused a parallel and rightward shift in the NPY concentration-response curve without depressing the maximal response. The contractile effect of NPY was potently inhibited in a competitive manner. The pA2 value for BIBP 3226 was 7.01 +/- 0.08, a value equivalent to that observed in the rabbit saphenous vein. Although increasing concentrations of BIBP 3226 shifted the concentration-response curve of PYY to the right without any significant decrease in the maximal vasoconstrictor response, the antagonism appeared non-competitive as the slope of the Schild plot was significantly different from unity (0.58 +/- 0.04). 4. In conclusion, these data confirm that BIBP 3226 is a potent and selective nonpeptide Y1 receptor antagonist. Moreover, they show that complex interactions occur between BIBP 3226 and postjunctional receptors activated by PYY. We postulate that BIBP 3226 might discriminate between the effects of NPY and PYY at the postjunctional level in the rat tail artery. It may be that distinct receptors for NPY and PYY exist; these may or may not allosterically interact with each other. Another working hypothesis would be that there is a single receptor complex with allosterically interacting binding sites for the two peptides.     

Changes in hypothalamic neuropeptide Y concentrations induced by cholecystokinin analogues

Neuropeptide Y (NPY) and cholecystokinin (CCK) are two peptides involved in opposite ways in the control of food intake. A possible interaction between NPY and CCK has not yet been well defined. Two CCK derivatives with agonistic and antagonistic properties were studied with regard to their effects on brain and plasma NPY levels. The CCK agonist decreased NPY levels in plasma and in the hypothalamus but not in the other brain areas assayed. The CCK antagonist reversed the agonist-induced decrease in both plasma and hypothalamus. These results suggest a negative relation between NPY and CCK peptides, which is not surprising given their opposite role in feeding regulation. The hypothalamus, a preferential site of this regulation, appears to be the brain area most involved in the NPY-CCK interaction. The plasma NPY level variations closely reflect the hypothalamic profile, suggesting a direct release of NPY by a mechanism that remains to be investigated.     

Effects of the neuropeptide Y Y(2) receptor antagonist BIIE0246 on presynaptic inhibition by neuropeptide Y in rat hippocampal slices

We previously reported that (S)-N(2)-[[1-[2-[4-[(R,S)-5, 11-dihydro-6(6h)-oxodibenz[b, e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cylopentyl]a cetyl]-N-[2-[1, 2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2, 4-triazol-4-yl]ethyl]argininamid, BIIE0246, is a potent and highly selective neuropeptide Y Y(2) receptor antagonist. Neuropeptide Y Y(2) receptors have been proposed to mediate the inhibition by neuropeptide Y of excitatory synaptic transmission in rat hippocampus. Therefore, we investigated the effects of BIIE0246 on the electrophysiological properties of neuropeptide Y in rat hippocampal slices and determined the affinity of this novel antagonist for rat hippocampal neuropeptide Y Y(2) receptors. BIIE0246 displayed an affinity of IC(50)=4.0+/-1.6 (n=4) for neuropeptide Y receptor binding sites labelled by 125I-neuropeptide Y in rat hippocampal membranes. At a concentration of 1 microM, BIIE0246 completely antagonized the inhibitory effects of 300 nM neuropeptide Y on synaptic transmission in rat hippocampal slices. This is the first study showing that a selective neuropeptide Y Y(2) receptor antagonist is able to block neuropeptide Y mediated effects in the hippocampus and unambiguously characterizes the presynaptic receptor in the rat hippocampus as the neuropeptide Y Y(2) receptor.     

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.

     

Ligand internalization by cloned neuropeptide Y Y5 receptors excludes Y2 and Y4 receptor-selective peptides

In human embryonic kidney-293 (HEK-293) cells, the cloned human neuropeptide Y Y5 receptor saturably internalized agonists, with the rank order of neuropeptide Y-(19-23)-[Gly1,Ser3,Gln4,Thr6,Ala31,Aib32,Gln34]human pancreatic polypeptide (neuropeptide Y-Aib-pancreatic polypeptide)>human neuropeptide Y>porcine peptide YY>[Pro34]human peptide YY>[Leu31,Pro34]human peptide YY>human peptide YY-(3-36). Human pancreatic polypeptide competed [125I]neuropeptide Y binding and internalization in neuropeptide Y Y5 receptor-expressing cells, but itself showed no internalization. The internalization was strongly dependent on temperature. The surface binding, and especially the internalization, of human neuropeptide Y were highly sensitive to the clathrin network inhibitor phenylarsine oxide, and to the cholesterol-complexing antibiotic filipin III. The internalized ligands were present in particles corresponding to secondary endosomes in Percoll gradients, but especially in particles banding with the acid hexosaminidase lysosomal marker. At any temperature tested, internalization of the neuropeptide Y Y5 receptor driven by human neuropeptide Y in HEK-293 cells was much slower than the internalization of the neuropeptide Y Y1 receptor reported in the same cells, or in Chinese hamster ovary (CHO) cells. The neuropeptide Y Y5 receptor subtype could be the metabotropic receptor responding to protracted challenges by neuropeptide Y-like peptides, and its density could be little sensitive to concentration of extracellular agonists.     

Probing the functional conformation of neuropeptide Y through the design and study of cyclic analogues.

The functional importance of the PP-fold conformation in neuropeptide Y (NPY) was investigated. NPY and N alpha-Ac-NPY(10-36), and corresponding cyclic analogues cyclo18,22-[Lys18,Asp22]-NPY and N alpha-Ac-cyclo18,22-[Lys18,Asp22]-NPY(10-36), were synthesized. Strategies for synthesis of the cyclic analogues included the use of the Kaiser oxime resin and a segment condensation approach. Circular dichroism studies in phosphate buffer, pH 5.0, indicated self-association of all four peptides at low micromolar concentrations. Monomeric N alpha-Ac-NPY(10-36) showed only 13% alpha-helix, compared to 32% alpha-helix for monomeric NPY, demonstrating a helix-stabilizing effect of residues 1-9 that is consistent with the PP fold. The [Lys18,Lys22] lactam bridge stabilized the helical conformation in N alpha-Ac-NPY(10-36)(51% alpha-helix), but was helix destabilizing in NPY (21% alpha-helix). In rat brain receptor binding assays, the cyclic and linear N alpha-Ac-NPY(10-36) analogues were equipotent (IC50 = 13 nM for 125I-BH-NPY displacement), although the cyclic analogue was twice as potent in rat vas deferens assays. NPY was more potent than its cyclic analogue in the brain receptor binding assays (IC50 = 0.07 and 0.25 nM, respectively), but these peptides were equipotent in the vas deferens assays. These results support a functional role for the PP fold in NPY and correlate with the solution conformations of the monomeric peptides.     

Modulation by neuropeptide Y of parasympathetic nerve-evoked nasal vasodilatation via Y2 prejunctional receptor.

1. In pentobarbitone anaesthetized dogs, preganglionic stimulation of the superior cervical sympathetic nerve (15V, 1 ms, 10 Hz) induced marked reduction of nasal arterial blood flow, whereas parasympathetic nerve stimulation (5 V, 1 ms, 10-30 Hz) evoked frequency-dependent vasodilatation. 2. Sympathetic nerve stimulation for 3 min at 10 Hz evoked significant (P < 0.05) and prolonged attenuation of the vasodilator response to subsequent parasympathetic stimulation. Pretreatment with phentolamine (0.5 mg kg-1 h-1), propranolol (1 mg kg-1) and atropine (0.5 mg kg-1) reduced the vasoconstrictor effect of sympathetic stimulation by 35 +/- 4% whereas the parasympathetic nerve-evoked vasodilatation was not significantly modified. Atropine-resistant parasympathetic vasodilatation remained significantly attenuated for more than 30 min after non-adrenergic sympathetic nerve-evoked vasoconstriction. 3. Vasodilator effects of exogenous vasoactive intestinal polypeptide and peptide histidine isoleucine and vasoconstrictor effects of exogenous neuropeptide Y (NPY) and the NPY analogue [Leu31, Pro34] NPY (Y1-receptor agonist, 8 nmol kg-1), were not altered by adrenoceptor antagonists and atropine f1p4eas the effects of exogenous noradrenaline and acetylcholine were virtually abolished. Attenuation of parasympathetic-evoked vasodilatation could be mimicked by exogenous NPY (8 nmol kg-1) and the NPY analogue, N-acetyl [Leu28, Leu31] NPY 24-36 (Y2-receptor agonist, 20 nmol kg-1) but not by exogenous Y1-receptor agonist. The Y2-receptor agonist did not show significant vasoconstrictor action. 4. It is concluded that sympathetic nerve stimulation attenuates parasympathetic vasodilatation via NPY release acting on prejunctional Y2 receptors.     

Autoinhibitory function of the sympathetic prejunctional neuropeptide Y Y(2) receptor evidenced by BIIE0246

The significance of neuropeptide Y Y(2) receptors in sympathetic nonadrenergic transmission was investigated using the novel selective antagonist BIIE0246 ((S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5 (4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide). In anaesthetized pigs pretreated with reserpine, and after transection of sympathetic nerves (depleted of noradrenaline), electrical stimulation of renal and splanchnic sympathetic nerves evoked vasoconstriction in, and overflow of neuropeptide Y-like immunoreactivity from, kidney and spleen, respectively. In the presence of BIIE0246, the nerve-evoked overflows of neuropeptide Y-like immunoreactivity were markedly increased and the splenic vasoconstrictor response prolonged. In addition, BIIE0246 caused splenic vasodilatation per se in this model where basal levels of circulating neuropeptide Y exceed 40 pM. It is concluded that endogenous neurogenical neuropeptide Y regulates its own release via activation of sympathetic prejunctional inhibitory neuropeptide Y Y(2) receptors in both spleen and kidney in the reserpinized pig. Moreover, when circulating levels of neuropeptide Y are moderately increased, activation of neuropeptide Y Y(2) receptors seems to contribute to basal splenic vascular tone.     

Amygdalar neuropeptide Y Y1 receptors mediate the anxiolytic-like actions of neuropeptide Y in the social interaction test

The effects of intra-amygdalar neuropeptide Y infusions were assessed in rats using the social interaction test. Neuropeptide Y administered into the central nucleus of the amygdala did not alter behavior, while injections into the basolateral nucleus of the amygdala produced an increased social interaction time. Furthermore, the anxiolytic-like effect was antagonized by co-administration of the potent neuropeptide Y Y1 receptor antagonist ((R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphen ylacetyl)-argininamide trifluoroacetate) 3304, but not with the inactive enantiomer ((R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphen ylacetyl)-argininamide trifluoroacetate) 3457. Therefore, neuropeptide Y produces an anxiolytic-like effect in the social interaction test through neuropetide Y Y1 receptors located in the basolateral amygdala.     

Preparation of fluorescent nonpeptidic neuropeptide Y receptor ligands: analogues of the quinazoline-type anti-obesity Y5 antagonist CGP 71683A

As part of a programme to develop fluorescence-based methods for the study of the interactions between G-protein coupled receptors (GPCRs) and their ligands the preparation of low molecular weight fluorescence-labelled neuropeptide Y (NPY) Y(5) antagonists is reported. The naphthylsulfonyl group in the potent quinazoline-type NPY Y(5) receptor antagonist CGP 71683A was replaced with a dansyl, nitrobenzoxadiazole (NBD) or acridine-9-carbonyl group. In radioligand binding studies on human Y(5) receptor expressing HEC-1B cells the substances labelled with acridine (K(i) 311 nM) and NBD (K(i) > 1000 nM) proved to be moderately active or inactive, respectively. By contrast, a K(i) value of 49 nM was found for the dansyl analogue compared to 2 nM for CGP 71683A. No binding to Y(1) receptors (SK-N-MC cells, displacement of [(3)H]propionyl-NPY) was detected for the new compounds at concentrations 相似文献   

Reduction of vasoconstriction mediated by neuropeptide Y Y2 receptors in arterioles of the guinea-pig small intestine.

Brief applications of a high-K+ solution were used to evoke transient constrictions of arterioles from the guinea-pig small intestine. Analogues of neuropeptide Y (NPY) selective for Y2-receptors reduced the constrictions, whereas NPY or a Y1-selective analogue potentiated the constrictions. We conclude that arteriolar smooth muscle has both Y1 and Y2 receptors, and suggest that Y2 receptors inhibit vasoconstriction by modulating the opening of voltage-sensitive Ca2+ channels. This may be related to the role of NPY that is present in some vasodilator nerves.     

Importance of a N-terminal aspartate in the internalization of the neuropeptide Y Y2 receptor

With human neuropeptide Y Y(2) receptor expressed in the Chinese hamster ovary (CHO) cells, the Asp(35)Ala mutation, and especially the change of Pro(34)Asp(35) to Ala(34)Ala(35), decrease the compartmentalization and strongly accelerate internalization of the receptor. These changes are not associated with alterations in agonist affinity, G-protein interaction, dimerization, or level of expression of the mutated receptors relative to the wildtype receptor. The proline-flanked aspartate in the N-terminal extracellular segment of the neuropeptide Y Y(2) receptor thus apparently has a large role in anchoring and compartmentalization of the receptor. However, the Pro(34)Ala mutation does not significantly affect the embedding and cycling of the receptor.     

Novel sst2-selective somatostatin agonists. Three-dimensional consensus structure by NMR

The 3D NMR structures of six octapeptide agonist analogues of somatostatin (SRIF) in the free form are described. These analogues, with the basic sequence H-DPhe/Phe2-c[Cys3-Xxx7-DTrp8-Lys9-Thr10-Cys14]-Thr-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Ala/Aph, exhibit potent and highly selective binding to human SRIF type 2 (sst2) receptors. The backbone of these sst2-selective analogues have the usual type-II' beta-turn reported in the literature for sst2/3/5-subtype-selective analogues. Correlating the biological results and NMR studies led to the identification of the side chains of DPhe2, DTrp8, and Lys9 as the necessary components of the sst2 pharmacophore. This is the first study to show that the aromatic ring at position 7 (Phe7) is not critical for sst2 binding and that it plays an important role in sst3 and sst5 binding. This pharmacophore is, therefore, different from that proposed by others for sst2/3/5 analogues.     

Central control of feeding behavior by neuropeptide Y

Obesity is a serious health problem in the Western societies, therefore its treatment has become the subject of intense interest in the scientific community. A significant number of recent publications enlist different central and peripheral factors which play important roles in the regulation of food intake, body weight and energy expenditure. Neuropeptide Y, a 36 amino acid peptide, which is quite abundant in the brain, seems to be one of the more important players in these regulations. Recently five NPY receptors have been cloned and pharmacological evidence strongly supports the existence of a sixth receptor. There are many contradictory findings regarding which NPY receptor mediates the effect of NPY on food intake. This article will review the effects of NPY on the regulation of food intake and energy expenditure and will discuss the pharmacological and molecular evidence as to which NPY receptor(s) mediate this effect. The review will also summarize the progress which has been made in the design of novel NPY-ergic ligands, especially NPY receptor antagonists, for potential use in the treatment of obesity.     

Vasoconstrictor effects of various neuropeptide Y analogues on the rat tail artery in the presence of phenylephrine.

1. The increase in perfusion pressure induced by neuropeptide Y (NPY), peptide YY (PYY) and related peptides were compared in the perfused rat tail artery precontracted by a submaximal concentration (1 microM) of the vasoconstrictor, phenylephrine. 2. NPY, PYY, [Leu31,Pro34]NPY, [Glu16,Ser18,Ala22,Leu28,31]NPY (ESALL-NPY) and the centrally truncated and stabilized analogues [D-Cys5,8-aminooctanoic acid7-20, Cys24]-NPY (D-Cys5-NPY) and [D-Cys7, 8-aminooctanoic acid8-17,Cys20]-NPY (D-Cys7-NPY) produced a concentration-dependent enhancement of the vasoconstrictor response induced by 1 microM phenylephrine. PYY was two times more potent than NPY and [Leu31,Pro34]NPY while ESALL-NPY, D-Cys7-NPY and D-Cys5-NPY were approximately 3, 5 and 16 times less potent than NPY respectively. NPY, D-Cys5-NPY and D-Cys7-NPY gave similar maximal responses whereas those observed for PYY, [Leu31,Pro34]NPY and ESALL-NPY were much greater than that of NPY. 3. NPY 13-36 and [des-Ser3,Lys4,Cys2,8-aminooctanoic acid3-24, D-Cys27]-NPY ([es-Ser3,Lys4]Cys2-NPY) were practically inactive at concentrations up to 3 microM, whereas [des-Ser3,Lys4,D-Cys2,8-aminooctanoic acid3-24,Cys27]-NPY ([des-Ser3,Lys4]D-Cys2-NPY), which differs from [des-Ser3,Lys4]Cys2-NPY in the disulphide bridge (a D-Cys in position 2 for [des-Ser3,Lys4]D-Cys2-NPY instead of an L-CYs for [des-Ser3,Lys4]Cys2-NPY) was a weak agonist the maximal effect of which could not be ascertained. 4. The contractile effects of [des-Ser3,Lys4]D-Cys2-NPY were additive with those of NPY and [Leu31,Pro34]NPY demonstrating that it is not a partial agonist but may simply not interact competitively with the receptor binding site for NPY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preferential expression of the neuropeptide Y Y1 over the Y2 receptor subtype in cultured hippocampal neurones and cloning of the rat Y2 receptor

1. Neuropeptide Y (NPY) and NPY receptors are most abundant in the hippocampal formation where they modulate cognitive functions. Expression of NPY receptors in rat cultured primary hippocampal cells was investigated in the present study by use of combined molecular, pharmacological and immunohistochemical approaches, including the cloning of the rat Y₂ receptor described here for the first time.
2. More than 70% of the hippocampal neurones were endowed with [¹²⁵I]-[Leu³¹,Pro³⁴]PYY Y₁-like receptor silver grain accumulations and Y₁ receptor immunostaining. These radio- and immuno-labelling signals were distributed over cell bodies and processes of bipolar, stellate and pyramidal-like neuronal cells, as confirmed by neurone-specific enolase and MAP-2 staining.
3. Competition binding profiles revealed that specific [¹²⁵I]-[Leu³¹,Pro³⁴]PYY binding was competitively displaced according to a ligand selectivity pattern prototypical of the Y₁ receptor sub-type with [Leu³¹,Pro³⁴]substituted NPY/PYY analogues>>C-terminal fragments=pancreatic polypeptides, with the non-peptide antagonist BIBP3226 being most potent. This profile excludes the possible labelling by [¹²⁵I]-[Leu³¹,Pro³⁴]PYY of the newly cloned Y₄, Y₅ and Y₆ receptors.
4. The expression of the genuine Y₁ receptor was confirmed by RT–PCR in hippocampal cultures. In contrast, negligible levels of Y₂-like/[¹²⁵I]-PYY_3–36 binding were detected in these cultures in spite of the presence of its mRNA, as characterized by RT–PCR. The expression of both the Y₁ and the Y₂ receptor mRNAs was also noted in normal embryonic hippocampal tissues showing that signals expressed in cultured neurones were also present in utero.
5. Taken together, these results suggest that the Y₁ receptor subtype may be of critical importance in the normal functioning of the rat hippocampus, especially during brain development and maturation.

     

Truncated, branched, and/or cyclic analogues of neuropeptide Y: importance of the pancreatic peptide fold in the design of specific Y2 receptor ligands.

Truncated, branched, and/or cyclic neuropeptide Y (NPY) analogues were tested for their ability to bind to the neuroblastoma cells, SK-N-MC (Y1 receptor) and SK-N-BE(2) (Y2 receptor). The design of such analogues was inspired by models of NPY based on the crystal structure of avian pancreatic polypeptide. The minimum length of the backbone was investigated using the following truncated analogues [binding affinity (nM) for Y1 and Y2 receptor subtypes respectively are given in parentheses]: des-AA10-17[D-Ala9]NPY (100, 0.9), des-AA7-23[D-Ala6]NPY (> 1000, 1.2), des-AA4-26[D-Ala3]NPY (> 1000, 120), cyclo(7,20)-des-AA10-17[Glu7,D- Ala9,D-Dpr20]NPY (100, nd), cyclo(2,27)-des-AA7-23[Glu2,D-Ala6,D-Dpr27]NPY (> 1000, 3.6), cyclo(2,30)- des-AA7-23[Glu2,D-Ala6,-D-Dpr30]NPY (> 1000, nd), cyclo(1,30)-des-AA4-26[Glu1,D-Ala3,D-Dpr30]NPY (> 1000, > 1000). A new family of branched NPY analogues corresponding to the partial deletion of the polyproline helix with conservation of the N-terminus was also examined: des-AA7-23[(Ac-NPY14-22)-epsilon-D-Lys6]NPY (> 1000, 2.1), des-AA7-23[(Ac-NPY7-22)-epsilon-D-Lys6]NPY (> 1000, 5.1), des-AA7-23-[(Ac-LEALEG-NPY14-22)-epsilon-D-Lys6]NPY (> 1000, 4.8). Finally, the role played by the flexible tail (residues 32-36) was studied with the following cyclic analogues: cyclo(30,34)-[Lys30,Glu34]NPY18-36 (> 1000, 360), cyclo(30,34)-[Orn30,Gly34]NPY18-36 (> 1000, 950), cyclo(30,34)-[Dpr30,Glu34]NPY18-36 (> 1000, 590), cyclo(33,36)-[Lys33,Glu36]NPY (> 1000, > 1000), cyclo(33,36)-[Lys33,Glu36]NPY18-36 (> 1000, > 1000). These results suggest that the Y1 receptor is highly discriminatory since deletion of residues 10-17, shown to have little effect on Y2 binding affinity, reduces Y1 affinity 50-fold. Bridging sites and constructs have been identified that may serve as useful leads in the design of more potent and selective analogues. We have identified two positions (9 and 6) where the introduction of a D amino acid is not detrimental to binding affinity. Whether this modification leads to the stabilization of a yet unidentified turn compatible with high Y2 receptor affinity will have to be determined by spectroscopic methods. Finally, stabilizing a putative alpha-helical conformation of the C-terminal heptapeptide of NPY18-36 has a deleterious effect on the Y1 and Y2 receptors.     

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.