Structural study of the N-terminal segment of neuropeptide tyrosine

A series of analogues of neuropeptide tyrosine (NPY) was synthesized by solid-phase peptide synthesis using BOP as a coupling reagent for the complete synthesis. A structure-activity study of the N-terminal portion of the molecule was performed with the analogues obtained by the successive replacement of the first 10 amino acids by the residue L-alanine. NPY and its analogues [Ala1-10]hNPY were tested for their potency on rat vas deferens and for their affinity to central nervous system receptors on a rat brain membrane preparation. The results suggest that the hypothetical polyproline type II helix structure of the N-terminal segment is involved in both potency and affinity. Indeed, the substitution by L-Ala of proline residues in position 2, 5, or 8 showed important losses of activity and affinity. The more important losses were observed with the replacement of Pro-5 or Pro-8. A critical loss of potency of hNPY was also observed after the substitution of the Tyr-1 residue by L-Ala, thus confirming the important role played by this residue for the full expression of the biological activity of NPY.     

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.     

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.     

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.     

Synthesis and characterization of a selective peptide antagonist of neuropeptide Y vascular postsynaptic receptors.

1. A cyclic dimeric nonapeptide neuropeptide Y (NPY) receptor antagonist, 1229U91, was synthesized by Fmoc chemistry and dimerised in solution. Its effects were assayed in mesenteric arteries from rats and mice, and in rat vas deferens. 2. Mesenteric arteries were cannulated and pressurised to 55 mmHg and the external diameters continuously measured. NPY, PYY, Leu31Pro34NPY and NPY(13-36) each caused concentration-related contractions with the order of potency PYY > or = Leu31Pro34NPY = NPY > NPY (13-36), consistent with the Y1 receptor subtype. 3. 1229U91 had no agonist activity in the arteries but caused a concentration-related rightward shift of NPY (mouse arteries) or Leu31Pro34NPY (rat) concentration-response curves. The antagonism was competitive with pKBS of 7.69 +/- 0.15 and 7.47 +/- 0.13 in the mouse and rat arteries, respectively. 4. Sympathetic nerves in the vas deferens were stimulated with a single electrical field pulse every 20 s and the twitch responses recorded. NPY, PYY, Leu31Pro34NPY and NPY(13-36) inhibited the twitches with the order of potency PYY > NPY > NPY(13-36) >> Leu31Pro34NPY, consistent with the Y2 receptor subtype. 5. 1229U91 inhibited the vas deferens twitch with a shallow concentration-response curve and a time-course of inhibition distinct from that of NPY. 1229U91 (30 microM) did not cause a rightward shift of the NPY concentration-response curve. 1229U91 is at least 5 orders of magnitude less potent in the vas deferens than in rat brain Y2 binding assays reported by others, suggesting that the brain and vas deferens Y2 receptors are different. 6. It is concluded that 1229U91 is a competitive antagonist of NPY Y1 vascular receptors and has additional properties that inhibit the electrically evoked twitch of the rat vas deferens.     

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.     

Failure of the putative neuropeptide Y antagonists, benextramine and PYX-2, to inhibit Y2 receptors in rat isolated prostatic vas deferens.

1. The pharmacological activity of neuropeptide Y (NPY) and some analogues in inhibiting the twitch contractions induced by electrical stimulation (single pulses at 25 V, 0.15 Hz, 1 ms) in the prostatic portion of the rat isolated vas deferens was investigated. The rank order of agonist potency was: PYY > NPY2-36 > NPY >> NPY13-36 >> NPY18-36 >> [Leu31,Pro34]NPY = hPP, which is consistent with the activation of a Y2 receptor. 2. The putative Y1 and Y2 antagonist, benextramine (BXT), incubated at 100 microM for 10 or 60 min, was ineffective against PYY-induced inhibition of the twitch response, suggesting that the prejunctional Y2 receptor in this tissue is different from the postjunctional one reported in the literature to be sensitive to BXT blockade. 3. The putative NPY antagonist, PYX-2, incubated at 1 microM for 20 min, was completely ineffective in antagonizing PYY-induced inhibition of twitches. 4. The twitch response was totally inhibited by suramin (100 microM) but was little affected by prazosin (1 microM). Furthermore, NPY was without effect on the dose-response curve to ATP in resting conditions. Taken together, these results suggest that in our paradigm, NPY inhibits the release of a purinergic neurotransmitter which mediates contraction of the prostatic portion of the rat vas deferens.     

BIIE0246, a potent and highly selective non-peptide neuropeptide Y Y(2) receptor antagonist

1. BIIE0246, a newly synthesized non-peptide neuropeptide Y (NPY) Y(2) receptor antagonist, was able to compete with high affinity (8 to 15 nM) for specific [(125)I]PYY(3 - 36) binding sites in HEK293 cells transfected with the rat Y(2) receptor cDNA, and in rat brain and human frontal cortex membrane homogenates. 2. Interestingly, in rat brain homogenates while NPY, C2-NPY and PYY(3 - 36) inhibited all specific [(125)I]PYY(3 - 36) labelling, BIIE0246 failed to compete for all specific binding suggesting that [(125)I]PYY(3 - 36) recognized, in addition to the Y(2) subtype, another population of specific NPY binding sites, most likely the Y(5) receptor. 3. Quantitative receptor autoradiographic data confirmed the presence of [(125)I]PYY(3 - 36)/BIIE0246-sensitive (Y(2)) and-insensitive (Y(5)) binding sites in the rat brain as well as in the marmoset monkey and human hippocampal formation. 4. In the rat vas deferens and dog saphenous vein (two prototypical Y(2) bioassays), BIIE0246 induced parallel shifts to the right of NPY concentration-response curves with pA(2) values of 8.1 and 8.6, respectively. In the rat colon (a Y(2)/Y(4) bioassay), BIIE0246 (1 microM) completely blocked the contraction induced by PYY(3 - 36), but not that of [Leu(31), Pro(34)]NPY (a Y(1), Y(4) and Y(5) agonist) and hPP (a Y(4) and Y(5) agonist). Additionally, BIIE0246 failed to alter the contractile effects of NPY in prototypical Y(1) in vitro bioassays. 5. Taken together, these results demonstrate that BIIE0246 is a highly potent, high affinity antagonist selective for the Y(2) receptor subtype. It should prove most useful to establish further the functional role of the Y(2) receptor in the organism.     

BODIPY-conjugated neuropeptide Y ligands: new fluorescent tools to tag Y1, Y2, Y4 and Y5 receptor subtypes

N-terminal labelled fluorescent BODIPY-NPY peptide analogues were tested in Y1, Y2, Y4 and Y5 receptor-binding assays performed in rat brain membrane preparations and HEK293 cells expressing the rat Y1, Y2, Y4 and Y5 receptors. BODIPY TMR/FL-[Leu31, Pro34]NPY/PYY were able to compete for specific [125][Leu31, Pro34]PYY-binding sites with an affinity similar to that observed for the native peptide at the Y1 (Ki=1-6 nM), Y2 (Ki>1000 nM), Y4 (Ki=10 nM) and Y5 (Ki=1-4 nM) receptor subtypes. BODIPY FL-PYY(3-36) was able to compete for specific Y2 (Ki=10 nM) and Y5 (Ki=30 nM) binding sites, but had almost no affinity in Y1 and Y4 assays. BODIPY FL-hPP was able to compete with high affinity (Ki; 1 and 15 nM) only in Y4 and Y5 receptor-binding assays. BODIPY TMR-[cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP and BODIPY TMR-[hPP(1-17), Ala31, Aib32]NPY were potent competitors only on specific Y5-binding sites (Ki=0.1-0.6 nM). As expected, these fluorescent peptides inhibited forskolin-induced cAMP accumulation, demonstrating that they retained their agonist properties. When tested in confocal microscopy imaging, fluorescent Y1 and Y5 agonists internalized in a time-dependent manner in Y1 and Y5 transfected cells, respectively. These results demonstrate that BODIPY-conjugated NPY analogues retain their selectivity, affinity and agonist properties for the Y1, Y2, Y4 and Y5 receptor subtypes, respectively. Thus, they represent novel tools to study and visualize NPY receptors in living cells.     

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.     

C-terminal neuropeptide Y fragments are mast cell-dependent vasodepressor agents.

Neuropeptide Y (NPY) is a well-established vasopressor agent present in sympathetic perivascular nerves. Recently, it was found that high doses of the peptide cause a biphasic pressor-depressor response upon intravenous administration. We now report that C-terminal NPY fragments (NPY-(18-36) and NPY-(22-36] given intravenously to conscious or pithed (areflexive) male Sprague-Dawley rats mimic the depressor component of the NPY-(1-36) response while displaying very low pressor activity. Additionally, we have found that the depressor component is blocked by the histamine H1-antagonist, mepyramine. Since the fragment, NPY-(22-36), was equipotent with NPY in inducing histamine release from isolated peritoneal mast cells, we conclude that short C-terminal NPY fragments, like NPY itself, act on mast cells to initiate histamine-mediated cardiovascular actions. Such actions may conceivably be accounted for by the abundance of positively charged amino acid residues in the C-terminus. Moreover, these fragments have little affinity for vascular NPY receptors, as indicated by their poor ability to displace iodinated NPY or peptide YY (PYY) from specific binding sites on vascular smooth muscle cells derived from rat aorta. In conclusion, we propose that short C-terminal NPY fragments, which contain several positively charged amino acid residues, retain the ability of NPY to release histamine from rat mast cells while being essentially devoid of direct vascular motor activity.     

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.     

Synthesis and hypertensive activity of neuropeptide Y fragments and analogues with modified N- or C-termini or D-substitutions

Porcine neuropeptide Y (NPY), NPY fragments, and analogues with D-Xaan, Ala9, D-Ala9, and Met17 substitutions or modifications to the C- or N-termini were synthesized. The synthesis and purification of these peptides was achieved by using routine laboratory strategies and techniques. The ability of these peptides to alter mean arterial pressure (MAP) and heart rate (HR) in conscious rats was monitored for 15 min following intraarterial administration. Potencies and efficacies of these peptides relative to NPY were determined by comparison of dose-response curves. Administration of 40 micrograms/kg NPY resulted in a rapid, though short-lived, rise in mean arterial pressure from a basal value of 107.0 +/- 2.6 to 157 +/- 5.5 mmHg (means +/- sem, n = 13). The ED50 (+/- SE) for this response was 3.04 +/- 0.88 micrograms/kg. Peptide YY (PYY) elicited a response that was similar in magnitude but with an ED50 (+/- SE, n = 3) of 0.76 +/- 0.24 micrograms/kg while porcine pancreatic polypeptide (pPP) was inactive when tested at 40 micrograms/kg (n = 4). Relative potencies for [Ac-Tyr1]NPY, [Ac-D-Tyr1]NPY, [des-amino-Tyr1] NPY, and [Me-Tyr1]NPY ranged from 1.1 to 2.2. Potencies relative to NPY for D-substitutions at positions 2-6 and 8-13 inclusive ranged from 0.1 to 1.0. Analogues with D-substitutions at positions 1-3 exhibited an extended duration of action. Analogues with D-substitutions at positions 33-35 inclusive were inactive at 40 micrograms/kg, and [D-Tyr36]NPY was 10-fold less potent than NPY, suggesting that the integrity of the C-terminal region is critical to the overall biological action of NPY. This conclusion is supported by studies with C- and N-terminal deletion peptides. NPY2-36 showed full intrinsic activity at 40 micrograms/kg and retains 40% of the hypertensive potency of NPY. There was a sequential decrease in efficacy upon further N-terminal deletion. In contrast to the finding with NPY2-36, modification of the C-terminus either from the native carboxamide to the free carboxylic acid or by deletion of the C-terminal residue resulted in analogues which were inactive at 40 micrograms/kg. These data indicate that an essentially full-length, C-terminally amidated NPY structure is required for the hypertensive activity observed in conscious rats upon intraarterial administration of NPY and NPY analogues.     

Structural requirements for neuropeptide Y18-36-evoked hypotension: a systematic study.

It has been shown that NPY and select C-terminal fragments of NPY that evoke a hypotensive response upon intraarterial administration in the rat also cause mast cell degranulation and histamine release in vitro. Additionally, elevation of plasma histamine levels has been observed concomitant with the hypotensive effect of NPY and various C-terminal fragments. In order to investigate whether the hypotensive response to NPY18-36 is correlated to this observed elevation of histamine in vivo, we sought to characterize the structural requirements for each activity. We conducted a systematic replacement of each amino acid in NPY18-36 by its D-isomer. Additionally, various modifications were made to the N- or C-terminii of NPY18-36. The following rank order of potency was obtained for the hypotensive action of these analogues of NPY18-36 relative to NPY18-36. Only one analogue ([D-Tyr21]NPY18-36) exhibited significantly enhanced potency. Eleven analogues of NPY18-36, ([D-Thr32]-, [D-Arg35]-, [D-Ile31]-, [D-Leu30]-, [D-Tyr27]-, [D-Ser22]-, [D-Tyr36]-, [D-Gln34]-, [D-Asn29]-, [D-Ala23]-, and [D-Arg33]NPY18-36) were equipotent with NPY18-36. Four analogues ([D-His26]-, [D-Ile28]-, and [D-Ala18]NPY18-36 and -NPY18-27) had reduced potency (10-80%) while eight analogues ([D-Arg19]-, [D-Tyr20], [D-Leu24]-, [D-Arg25]-, [Ac-Ala18]-, [Me-Ala18]-, [desamino-Ala18]NPY18-36 and NPY18-36 free acid) failed to produce a significant hypotensive response (less than 10%) at the doses tested. The sensitivity of NPY18-36 to chiral inversion of single residues or other modifications at the N-terminus suggested the presence of a conformationally well defined N-terminal pharmacophore. Additionally, five NPY18-36 analogues were tested for elevation of plasma histamine levels. The rank order of potency ([D-Thr32]NPY18-36 = [D-Tyr21]NPY18-36 much greater than NPY18-36 greater than [D-Ala18]NPY18-36 greater than [Ac-Ala18]NPY18-36) was correlated with each analogue's potency at evoking a hypotensive response. In contrast, NPY1-36 failed to evoke an elevation in plasma histamine levels despite its hypotensive effects. Hence, we conclude that the magnitude of the hypotensive response evoked by an NPY18-36 analogue is primarily a function of its ability to elevate plasma histamine levels. However, the mechanism underlying NPY1-36-evoked hypotension appears to be different.     

Differentiation between rat brain and mouse vas deferens delta opioid receptors

Certain enkephalin analogues, including those which contain the conformationally restricted amino acid E-(2R,3S)-cyclopropylphenylalanine [2R,3S)-delta E Phe), have been shown to have high affinity for brain delta opioid receptors but are much less active in mouse vas deferens bioassays. To investigate whether there are differences between delta opioid receptors in brain and mouse was deferens, the ability of a selective delta opioid compound, [D-Pen2,pCl-Phe4,D-Pen5]enkephalin (pCl-DPDPE), and [D-Ala2,(2R,3S)-delta E Phe4,Leu5]enkephalin methyl ester (CP-OMe), to inhibit [3H]pCl-DPDPE binding in both rat brain and mouse vas deferens were measured. pCl-DPDPE recognized brain and mouse vas deferens binding sites with equal affinity, however, CP-OMe showed 33 fold lower affinity in mouse vas deferens compared to brain. This suggests that mouse vas deferens delta opioid receptors may be distinct from brain delta opioid receptors.     

Age and castration modulate the inhibitory action of neuropeptide Y on neurotransmission in the rat vas deferens.

The potency of neuropeptide Y (NPY) to inhibit the electrically induced contractions of the epididymal half of the vas deferens diminishes markedly with age, being at least 20 times lower in the adult than in the 26-day-old rat. Castration sensitizes the epididymal segment to NPY in a testosterone-reversible manner. [Pro34]NPY was 3 times less potent than NPY in prepubertal rats and inactive in castrated adults, while NPY-(13-36) had no effect in either group. In the prostatic half, NPY and its analogs were active in rats from all ages studied; the order of potency being NPY greater than [Pro34]NPY greater than NPY-(13-36). The sensitivity of the prostatic segment from adult rats to NPY was unchanged by castration or testosterone replacement therapy. The NPY content of the ductus increases during development being higher in the prostatic than in the epididymal half at all ages studied. Castration decreases the peptide content in the two segments and the effect is prevented by testosterone administration. The present investigation demonstrated that the effect of NPY on vas deferens neurotransmission is subject to regulation by sex steroids, which affects differently the response of the two segments of the ductus.     

Effects of a selective neuropeptide Y Y2 receptor antagonist, BIIE0246, on Y2 receptors at peripheral neuroeffector junctions

1. This study investigated the effects of BIIE0246, a novel neuropeptide Y (NPY) Y2 receptor antagonist, on the inhibition of cholinergic neuroeffector transmission in rat heart and guinea-pig trachea and purinergic neuroeffector transmission in guinea-pig vas deferens produced by the NPY Y2 receptor agonist, N-acetyl [Leu28,31] NPY 24-36. 2. In pentobarbitone anaesthetized rats, supramaximal stimulation every 30 s, of the vagus nerve innervating the heart, increased pulse interval by approximately 100 ms. This response was attenuated by intravenous administration of N-acetyl [Leu28,31] NPY 24-36 (10 nmol x kg(-1)). 3. Transmural stimulation of segments of guinea-pig trachea at 1 min intervals with 5 s trains of stimuli at 0.5, 5, 10, 20 and 40 Hz evoked contractions which were reduced in force by N-acetyl [Leu28,31] NPY 24-36 (2 microM). 4. In guinea-pig vasa deferentia, the amplitude of excitatory junction potentials evoked by trains of 20 stimuli at 1 Hz was reduced in the presence of N-acetyl [Leu28,31] NPY 24-36 (1 microM). 5. In all preparations BIIE0246 attenuated the inhibitory effect of N-acetyl [Leu28,31] NPY 24-36 but had no effect when applied alone. 6. The findings support the view that the nerve terminals of postganglionic parasympathetic and sympathetic neurones possess neuropeptide Y Y2 receptors which, when activated, reduce neurotransmitter release.     

Structure-activity study of hCGRP8-37, a calcitonin gene-related peptide receptor antagonist.

A structure-activity study was carried out to determine the importance of the N-terminal amino acids of hCGRP8-37 in binding and antagonistic activity to CGRP receptors. Therefore, fragments of hCGRP8-37 as well as analogs obtained by the replacement of residues 9-12 by L-alanine were synthesized by solid-phase peptide synthesis, using BOP as a coupling reagent. The affinities of the peptides to CGRP receptors were evaluated in the rat brain, guinea pig atrium, and guinea pig vas deferens membrane preparations. Their antagonistic activities were measured in the guinea pig atria and rat vas deferens bioassays. The pharmacological characterization showed that arginine-11 and leucine-12 play a crucial role for the affinity of hCGRP8-37. Interestingly, it was observed that [Ala11]hCGRP8-37 was able to potentiate the twitch response of the electrically stimulated rat vas deferens. On the other hand, the substantial antagonistic potencies of analogs [Ala9]-, [Ala10]-, and [Ala12]hCGRP8-37, as compared to those of the fragments hCGRP10-37, hCGRP11-37, and hCGRP12-37, suggest that the side chains of Thr-9, His-10, and Leu-12 assume mainly a structural role. Accordingly, the conformational characterization of these peptides by circular dichroism spectroscopy revealed that the residues 9-12 are important for the integrity of the amphiphilic alpha-helix of hCGRP8-37.     

Adrenomedullin receptor binding sites in rat brain and peripheral tissues

The existence of specific adrenomedullin receptor binding sites was investigated using the agonist peptide fragment [125I]human adrenomedullin-(13-52) in rat brain, lung and vas deferens homogenates. Saturation-binding experiments suggest that [125I]human adrenomedullin-(13-52) binds to an apparent single population of sites with similar affinities (K(D) of 0.3 to 0.6 nM) but with different maximal binding capacity in the rat brain, lung and vas deferens homogenates (B(max) of 73, 1760 and 144 fmol/mg protein, respectively). Competition-binding experiments using various analogues and fragments of calcitonin gene-related peptide (CGRP) and adrenomedullin were also performed using this radioligand. Competition-binding profiles suggest the possible existence of heterogeneous populations of adrenomedullin receptor binding sites. For example, in rat brain, human adrenomedullin-(1-52) and human adrenomedullin-(13-52) competed against specific [125I]human adrenomedullin-(13-52) sites with competition curves best fitted to a two-site model. Additionally, human calcitonin gene-related peptide alpha (hCGRPalpha), [Cys(Et)(2,7)]hCGRPalpha and [[R-(R,(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide] (BIBN4096BS) competed against specific [125I]human adrenomedullin-(13-52) binding with profiles that were also best fitted to a two-site model. Furthermore, binding assays performed in the presence of GTPgammaS (100 microM) revealed that this compound inhibited 20% of specific [125I]human adrenomedullin-(13-52) sites in rat brain homogenates and competition curves of human adrenomedullin-(1-52) and [Cys(Et)(2,7)]hCGRPalpha against specific [125I]human adrenomedullin-(13-52) sites remained best fitted to a two-site model. Moreover, the existence of specific [125I]human adrenomedullin-(13-52) binding sites that are resistant to human adrenomedullin-(22-52) and human CGRP-(8-37) is suggested in the rat brain and vas deferens. Taken together, these data provide evidence for the possible existence of heterogeneous populations of adrenomedullin binding sites in rat brain and peripheral tissues.     

Characterisation of calcitonin gene-related peptide receptors in rat atrium and vas deferens: evidence for a [Cys(Et)(2, 7)]hCGRP-preferring receptor

The present study was performed in order to characterise calcitonin gene-related peptide (CGRP) receptor subtypes in rat left atrium and vas deferens by using [R-(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide (BIBN4096BS), a novel CGRP receptor antagonist. When CGRP was used as an agonist, BIBN4096BS exhibited an almost 10-fold higher affinity for CGRP receptors in rat left atrium compared to those in the vas deferens, indicating that CGRP acts through different CGRP receptor subtypes in these two tissues. In addition, BIBN4096BS was almost 10-fold more potent in antagonizing [Cys(Et)^2,7]hCGRP and human adrenomedullin-induced responses than CGRP-induced responses in rat vas deferens. This might indicate receptor heterogeneity in rat vas deferens. Accordingly, the present work provides first experimental evidence that the rat vas deferens contains two CGRP-like receptor subtypes. Namely, the CGRP₂ receptor and a “novel” receptor that possesses low efficacy for CGRP and that is selectively stimulated by [Cys(Et)^2,7]hCGRP or adrenomedullin and which can be blocked with high affinity by BIBN4096BS.