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.     

Comparative structural requirements of brain neuropeptide Y binding sites and vas deferens neuropeptide Y receptors

A series of fragments and analogues of neuropeptide Y (NPY), both human (hNPY) and porcine (pNPY), were synthesized and tested for their affinities at brain NPY receptor binding sites and their potencies in inhibiting the electrically stimulated twitch response of rat vas deferens. Results with N- and C-terminal fragments suggest that amino acid residues in the N-terminal portion of the molecule are mostly important for recognition of brain and vas deferens NPY receptors, in addition to being relevant for the maintenance of adequate receptor affinity. On the other hand, C-terminal amino acid residues appear to be responsible for triggering receptor activation in the rat vas deferens preparation, because full intrinsic activity is maintained with fragments up to NPY18-36. C-terminal fragment NPY25-36 and N-terminal fragment NPY1-15 were devoid of affinity for [3H]NPY brain receptor sites and showed no activity in the rat vas deferens preparation. Similarly, N-terminal fragment hNPY1-24CONH2 showed no affinity toward [3H]NPY brain receptor sites and no inhibition of the twitch response in the rat vas deferens preparation at concentrations up to 1.0 microM. On the contrary, this fragment appears to selectively increase the amplitude of the twitch response to electrical stimulation at low micromolar concentrations, an effect opposite to that of NPY and all other NPY fragments and analogues studied here. The exact mechanism mediating this contractile action of hNPY1-24CONH2 remains to be established. Modifications of the tyrosine residue in position 20 led to the development of two analogues, [D-Tyr20]hNPY and [D-Trp20]hNPY, which show an apparent preference for the vas deferens NPY receptor. On the other hand, substitutions of the tyrosine residue in position 21 by a phenylalanine ([Phe21]hNPY) or a methylated tyrosine residue ([Tyr-O-Me21]hNPY) produced analogues demonstrating an apparent preference for the brain receptor site. This suggests that modifications of tyrosine residues at positions 20 and/or 21 may eventually lead to the development of NPY analogues distinguishing between the most abundant class of sites present in the brain and vas deferens, respectively.     

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.     

Structure activity studies of mast cell activation and hypotension induced by neuropeptide Y (NPY), centrally truncated and C-terminal NPY analogues.

1. Neuropeptide-induced histamine release is thought to occur via receptor-independent mechanisms, with net charge and lipophilicity being important factors. 2. In this study, the histamine releasing ability of neuropeptide Y (NPY), two C-terminal segments of NPY and 13 centrally truncated NPY analogues was examined. These results were compared with the ability of the peptides to bind to the Y2 receptor in the rabbit kidney membrane model and with their hypotensive actions in the anaesthetized-rat model. 3. All analogues tested, with the exception of [Glu4,25,33,35]-NPY(1-4)-Ahx-(25-36) and [Asp4,25,33,35]NPY(1-4)-Ahx-(25-36) which were devoid of histamine releasing activity, evoked a dose-dependent histamine release but there were marked differences between the peptides. The native peptide was the least active. 4. Histamine release was not linked to the ability of the peptides to displace NPY from Y2 receptors. There was a statistical correlation between the hypotensive effects expressed as ED10 values (mumol kg-1, which induced a blood pressure decrease of 10 mmHg) and the EC25 for histamine release (r = 0.62, P = 0.04), although histamine release may not be the sole determinant of the alterations in blood pressure. 5. There was a strong negative correlation between EC25 for histamine release and net positive charge (r = -0.93, P = 5.7 x 10(-7), i.e. increasing the net positive charge caused greater histamine release. However, there was a 12 fold difference in activity amongst the most positively charged analogues (+5). Helicity did not correlate with histamine releasing ability. 6. In the development of NPY-related drugs the avoidance of compounds with net positive charge is recommended.     

Potent V2/V1a vasopressin antagonists with C-terminal ethylenediamine-linked retro-amino acids.

We report the solid-phase synthesis and antagonistic potencies of 25 analogues (1-25) of [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-O-ethyl-D-tyrosine,4-valine]arginine-vasopressin (d(CH2)5D-Tyr(Et)2-VAVP) (A) and of the related Ile4 (D) and [D-Phe2,Ile4] (E) analogues, potent antagonists of the antidiuretic (V2-receptor) and of the vasopressor (V1a-receptor) responses to arginine-vasopressin (AVP). Six of these peptides (1, 13, 17, 19, 21, and 23) have the Pro-Arg-Gly-NH2 tripeptide side chain fully or partially replaced or extended by ethylenediamine (Eda). The remaining 19 peptides have L- or D-amino acids retrolinked to these six C-terminal Eda peptides. Peptides 1, 13, 17, and 19 all have the ring structure of (A). Their side-chain structures are as follows: 1, Eda; 13, Pro-Eda; 17, Pro-Arg-Eda; 19, Arg-Gly-Eda. Peptide 21 is the Pro-Arg-Eda analogue of D; peptide 23 is the Pro-Arg-Gly-Eda analogue of E. Peptide 2 is the retro-Arg analogue of 1. Its side-chain structure is Eda<--Arg. Peptides 3-6 are analogues of 2 which have the D-Tyr-(Et)2 residue replaced by L-Tyr(Et)2 (3), D-Phe2 (4), D-Ile2 (5), or D-Leu2 (6), respectively. Peptides 7-12 are analogues of 2 which have the C-terminal retro-Arg replaced in retrofashion by D-Arg (7), Gly (8), Orn (9), D-Orn (10), D-Lys (11), or Arg-Arg (12). Peptides 14-16 have D-Orn (14), D-Lys (15), and D-Arg (16) retrosubstituted to peptide 13. Peptides 18, 20, and 22 are the retro-Arg-substituted analogues of 17, 19, and 21, respectively. Peptides 24 and 25 have Val and D-Val in retrolinkage with 23, respectively. All 25 peptides were examined for agonistic and antagonistic potencies in AVP V2/V1a assays. With the exception of peptides 5 and 6, all exhibit potent anti-V1a antagonism, with anti-V1a pA2 values in the range 7.64-8.33.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin on cholinergic and noradrenergic neurotransmission in isolated atria

In rabbit isolated atria, [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin (0.1-3 microM) inhibited responses to cholinergic nerve stimulation in a concentration-dependent manner without affecting responses to exogenous acetylcholine. The inhibitory effect was blocked by the opiate receptor antagonist naloxone (1 microM). In rabbit atria in which the transmitter acetylcholine stores had been radioactively labelled by preincubating the tissue in [3H]choline, tetrodotoxin (100 ng/ml) significantly (P less than 0.001) blocked the stimulation-induced (2 Hz for 3 min) release of radioactivity. Both [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin (0.3 and 1 microM) significantly decreased stimulation-induced radioactivity release and their effects were blocked by naloxone (1 microM). In rat isolated atria, [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin (0.3-3 microM) inhibited responses to cholinergic nerve stimulation without affecting responses to exogenous acetylcholine. The inhibitory effect was blocked by naloxone (1 microM). In guinea-pig isolated atria, responses to cholinergic nerve stimulation were unaffected by the enkephalin analogues. In rabbit, rat and guinea-pig isolated atria, responses to noradrenergic nerve stimulation and exogenous noradrenaline were unaffected by the enkephalin analogues.     

Structure-activity studies of antagonists of luteinizing hormone-releasing hormone with emphasis on the amino-terminal region

The structure-activity relationship of the hydrophobic amino terminal region of the antagonist [N-Ac-D-Nal1,D-pClPhe2,D-Trp3,D-Arg6,Phe7,D-Ala10]-LH- RH has been investigated by the incorporation of a variety of amino acids with emphasis on positions 1, 2, and 3. The analogues were prepared by routine solid-phase peptide synthesis. All purifications were performed in two stages: gel permeation chromatography followed by preparative, reversed-phase, high-performance chromatography. The analogues were assayed in a standard rat antiovulatory assay using a 40% propane-1,2-diol-saline vehicle. A simplified antagonist was developed that allowed the removal of the custom-synthesized D-pClPhe and the labile D-Trp while retaining antiovulatory potency. The compound [N-Ac-D-Nal1,D-Phe2,3,D-Arg6,Phe7,D-Ala10]-LH-RH caused a 56% blockade of ovulation at the 500-ng dose and is approximately equipotent with the parent analogue in this system.     

Effects of amino acid chirality and the chemical linker on the cell permeation characteristics of cyclic prodrugs of opioid peptides

Previously, our laboratory showed that the oxymethyl-modified coumarinic acid (OMCA) cyclic prodrug of the opioid peptide DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) exhibited low permeation across both the intestinal mucosa and the blood-brain barrier (BBB). This low cell permeation arose from its strong substrate activity for efflux transporters in these biological barriers. In an attempt to determine whether the chirality of the amino acid asymmetric centers could influence the solution structure of the cyclic prodrugs and thus their substrate activities for efflux transporters, we synthesized cyclic prodrugs of the opioid peptides H-Tyr-Ala-Gly-Phe-D-Leu-OH ([Ala2,D-Leu5]-Enk), H-Tyr-D-Ala-Gly-Phe-Leu-OH ([D-Ala2,Leu5]-Enk), and H-Tyr-Ala-Gly-Phe-Leu-OH ([Ala2,Leu5]-Enk). In an attempt to determine whether the chemical linker (OMCA) bestowed efflux substrate activity on the cyclic prodrugs, we synthesized capped linear derivatives (acetylated on the N-terminal and amidated on the C-terminal end) of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk. The solution conformations of the cyclic prodrugs were determined by molecular dynamics simulations using two-dimensional NMR data. The physicochemical properties (molecular surface area, polar surface area, and cLogP) were estimated computationally using Sybyl. Cell permeation characteristics were assessed using Caco-2 cells in the presence and absence of known inhibitors of efflux transporters. Despite apparent differences in their solution conformations and their physicochemical properties, the cyclic prodrugs of DADLE, [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk all exhibited strong substrate activity for efflux transporters in Caco-2 cells. In contrast, the capped linear derivatives of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk exhibited very poor substrate activity for efflux transporters in Caco-2 cells. Therefore, the substrate activities of the cyclic prodrugs for efflux transporters in Caco-2 cells and in the intestinal mucosa and the BBB in vivo are most likely due to the chemical linker used to prepare these molecules and/or its effect on solution structures of the prodrugs.     

Opiate-like peptides. Part VIII. Methylamides and dimethylamides of [D-Leu5]-enkephalin and [D-Ala2, D-Leu5]-enkephalin. Synthesis and analgesic activity

Syntheses of [D-Ala2, D-Leu5]-enkephalin, methyl ester of [D-Ala2, D-Leu3]-enkephalin, methylamides and dimethylamides of [D-Leu5]-enkephalin and [D-Ala2, D-Leu5]-enkephalin are described together with their analgesic activity determined on the basis of four analgesic tests: the hot-plate method, the reaction to electric stimulus, the tail immersion test and the frequency of writhing syndrome test. The neurotoxicity was estimated by the rota-rod test. The most pronounced analgesic effect was induced by compound: [D-Ala2, D-Leu5]-enkephalin, [D-Ala2, D-Leu5-OMe]-enkephalin and [D-Ala2, D-Leu5-NMe2]-enkephalin. In the tail immersion test all analogs did not exhibit analgesic activity.     

Characterization of vascular neuropeptide Y receptors.

1. In the present study we compared neuropeptide Y (NPY) and NPY-related analogues for their ability to activate or bind to vascular NPY receptors in four experimental set-ups. Previous results have suggested the existence of different receptor subtypes, Y1 receptors requiring full-length NPY (1-36) or [Pro34]-NPY, and Y2 receptors recognizing also N-terminally truncated forms of NPY but not [Pro34]-NPY. 2. NPY 1-36 and [Pro34]-NPY dose-dependently increased arterial pressure in the anaesthetized rat with a similar magnitude and potency. NPY 2-36 was much less potent than NPY 1-36. NPY 4-36 and NPY 11-36 were inactive even at a dose as high as 10 nmol kg-1. 3. NPY 1-36, [Pro34]-NPY, NPY 2-36 and NPY 5-36 concentration-dependently increased the coronary resistance in the Langendorff preparation of the rat. NPY 1-36 and [Pro34]-NPY were equipotent, while NPY 2-36 and NPY 5-36 were about 7 and 20 times less potent. At 0.3 microM, NPY 11-36, NPY 20-36 and NPY 22-36 induced a slight contraction while NPY 23-36 was inactive. 4. NPY 1-36, [Pro34]-NPY, NPY 2-36, NPY 4-36, NPY 5-36 and NPY 11-36 evoked concentration-dependent contractions in the isolated inferior caval vein of the rat and guinea-pig. [Pro34]-NPY was more potent than NPY 1-36. NPY 2-36 was equipotent with NPY 1-36, while NPY 4-36, NPY 5-36 and NPY 11-36 were approximately 30 times less potent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the CH2NH and CH2NAc peptide bond isosteres on the antagonistic and histamine releasing activities of a luteinizing hormone-releasing hormone analogue

The effect of the CH2NH and CH2NAc peptide bond isosteres on the antagonistic and histamine releasing activities of the LH-RH antagonist [N-Ac-D-Nal1,D-Phe2,3,D-Arg6,Phe7,D-Ala10] LH-RH was investigated. The moieties were introduced by facile, racemization-free solid-phase synthesis in an attempt to reduce the histamine releasing activity inherent to the most potent analogues while retaining high antiovulatory activity. The psi [CH2NH] isostere was incorporated at each CONH site with the exception of 8-9, which involves Pro, by reductive alkylation with a protected amino acid aldehyde in the presence of NaBH3CN during conventional solid-phase peptide synthesis. The psi [CH2NH] group was extremely resistant to derivatization and could only be partially acetylated to give psi [CH2NAc]. The analogues were cleaved from the resin with simultaneous deprotection by anhydrous hydrogen fluoride and purified to homogeneity in two stages: gel permeation followed by preparative reversed-phase liquid chromatography. The analogues were assayed in standard rat antiovulatory and in vitro histamine release assays. The isosteres caused a loss of the antiovulatory activity of the antagonist at the 50-microgram dose when incorporated at the positions 1-2, 2-3, 3-4, and 7-8. Incorporation at the other positions resulted in a less marked reduction in activity relative to the unmodified parent analogue. No significant effect was noted on the potent histamine releasing activity of the analogues.     

Structure-activity studies of neurotensin on muscular rigidity and tremors induced by 6-hydroxydopamine lesions in the posterolateral hypothalamus of the rat

It has previously been reported that intracerebroventricular administration of neurotensin (30 micrograms) reduced muscular rigidity and tremors, induced by a neurochemical lesion with 6-hydroxydopamine in the posterolateral hypothalamus of rats. In the present study, the effects of two fragments (NT1-10 and NT8-13) and two analogues ([D-Tyr11]-NT and [Ala11]-NT) of neurotensin on the grasping time (index of muscle rigidity) and tremors in 6-hydroxydopamine-lesioned rats are reported. Intracerebroventricular administration with 120 micrograms of NT1-10 and [Ala11]-NT had no effect on the muscle rigidity and tremors induced by the neurochemical lesion. The administration of NT8-13 60 micrograms) significantly attenuated both behavioural responses. The analogue [D-Tyr11]-NT produced a much greater attenuation of the muscle rigidity and tremors. The dose of 1.8 micrograms of [D-Tyr11]-NT significantly reduced the grasping time, while the number of tremors was attenuated with the threshold dose of 0.9 micrograms. Together, these results suggest that the effects of neurotensin on muscle rigidity and tremors, induced by pretreatment with 6-hydroxydopamine injected into the posterolateral hypothalamus, were not caused by non-specific effects but largely depended on the carboxy terminal of the peptide. The tyrosine residue in position 11 of the molecule plays a critical role in the action of neurotensin, as shown with the high potency and duration of action of the analogue [D-Tyr11]-NT. As previously suggested, the greater effect with [D-Tyr11]-NT may be due to greater resistance of the analogue to enzymatic degradation because of the incorporation of the D-Tyr amino acid, in position 11 of neurotensin.     

Discrimination by benextramine between the NPY-Y1 receptor subtypes present in rabbit isolated vas deferens and saphenous vein.

1. In order to characterize the neuropeptide Y (NPY) Y1 receptors known to be present in rabbit isolated vas deferens and saphenous vein, the pharmacological activity of the selective NPY Y1 receptor agonists, [Leu31,Pro34] NPY and various other peptide agonists, together with the putative NPY antagonist, benextramine, were compared in the two tissues. 2. In rabbit isolated saphenous vein, cumulative dose-response curves to various NPY agonists were obtained. All the peptides tested caused contractions which developed quite slowly. The rank order of potency obtained was: PYY > NPY > [Leu31,Pro34] NPY = NPY2-36 > hPP >> NPY13-36 = NPY18-36. Incubation with benextramine (BXT) at 100 microM for 30 min irreversibly abolished the contractile response to [Leu31,Pro34] NPY but was ineffective against NPY18-36-induced contractions. 3. Cumulative dose-response curves to [Leu31,Pro34] NPY were performed in the same preparation before and after incubation with 100 microM BXT for 20 min in order to inactivate NPY Y1 receptors. The pKA (-logKA) estimation for [Leu31,Pro34] NPY was 7.60 +/- 0.30 using the operational model and 7.20 +/- 0.33 using the null method; the difference between the two methods was not statistically significant (P = 0.36). 4. Prostatic segments of rabbit vas deferens were electrically stimulated with single pulses. Immediately after stabilization of the contractile response, a cumulative dose-response curve to various NPY agonists was obtained in each tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characterization of neuropeptide Y (NPY) receptors in human cerebral arteries with selective agonists and the new Y1 antagonist BIBP 3226.

1. We have characterized pharmacologically the receptor subtype(s) responsible for the neuropeptide Y (NPY)-induced vasoconstriction in human cerebral arteries. NPY, PYY and several of their derivatives with well defined affinities at the known Y1 and Y2 receptor subtypes were used. Moreover, we tested the ability of the new Y1 receptor antagonist, BIBP 3226, to antagonize the NPY-induced cerebral vasoconstriction. 2. NPY, PYY and their agonists with high affinities at the Y1 receptor subtype ([Leu31-Pro34]-NPY and [Leu31-Pro34]-PYY) elicited strong, long lasting and concentration-dependent contractions of human cerebral arteries. Compounds with Y2 affinity such as PYY3-36 or NPY13-36 either elicited a submaximal contraction at high concentrations or failed to induce any significant vasomotor response. Also, the application of NPY or the specific Y1 agonist, [Leu31-Pro34]-NPY, to human cerebral vessels pretreated with the Y1 agonist, NPY13-36, resulted in contractile responses identical to those obtained when these compounds were tested without prior application of NPY13-36. 3. The order of agonist potency at the human cerebrovascular receptor was: [Leu31-Pro34]-NPY = [Leu31-Pro34]-PYY > or = NPY > PYY > PYY3-36 > > > NPY13-36, which corresponded to that reported previously at the neuronal and vascular Y1 receptors. 4. Increasing concentrations (10(-9)-10(-6) M) of the Y1 receptor antagonist, BIBP 3226, to human cerebral vessels caused a parallel and rightward shift in the NPY dose-response curves without any significant change in the maximal contractile response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opiate-receptor interactions on single locus coeruleus neurones

Intracellular recordings were made from neurones of the rat locus coeruleus (LC) which were located in a slice of pons superfused in vitro. Opioid agonists and antagonists were applied by adding them to the superfusing solution; normorphine and enkephalin analogues were also applied by ejecting a few nanoliters of a solution which contained the drugs from a pipette situated above the tissue slice. Opioid agonists hyperpolarized LC neurones. This has been shown previously to result from an increase in the membrane potassium conductance. The lowest concentration of normorphine which was effective was 30 nM, the EC50 was 1 microM, and the maximum effect was observed with 30 microM. The irreversible antagonist beta-funaltrexamine (beta-FNA) was used to estimate the dissociation equilibrium constants; these ranged from 9-16 microM for normorphine and [Met5]enkephalin and was about 2 microM for [D-Ala2,D-Leu5]enkephalin. beta-FNA also blocked the hyperpolarization caused by [D-Ala2,D-Leu5]enkephalin, ethylketacyclazocine, and [D-Ser2,D-Leu5] enkephalin-Thr. Naloxone reversibly antagonized the hyperpolarizations caused by normorphine and [D-Ala2,D-Leu5]enkephalin, with a dissociation equilibrium constant of 2 nM. It is suggested that the opioid hyperpolarization of LC neurones is mediated by a receptor having a high affinity for naloxone, previously termed a mu-receptor. The affinity of this receptor for normorphine appears to be 3 to 4 orders or magnitude lower than its affinity for naloxone.     

Dermorphin analogues containing D-kyotorphin: structure-antinociceptive relationships in mice.

The antinociceptive effects of synthetic dermorphin and its analogues containing D-Arg in position 2 injected into the lateral cerebroventricle were examined in conscious mice. Intracerebroventricular (i.c.v.) administration of dermorphin and [D-Arg2] dermorphin produced potent and long-lasting antinociceptive activity as assayed by the tail-pressure test. Dermorphin and [D-Arg2] dermorphin were 210 and 52 times more potent than morphine, respectively. The antinociceptive effects produced by these heptapeptides were antagonized by a low dose (0.5 mg kg-1, i.p.) of the opioid antagonist naloxone. The ED50 values for [D-Arg2] dermorphin (1-6), (1-5) and (1-4) were not significantly different from that for [D-Arg2] dermorphin. The potency of the shortest fragment, [D-Arg2] dermorphin (1-2) was found to possess a severely reduced activity, whilst [D-Arg2] dermorphin (1-3) maintained activity and was 10 times more potent than morphine. [D-Arg2] dermorphin analogues showed almost identical effects when tested on the electrically-induced contractions of the guinea-pig isolated ileum. These results led us to conclude that the presence of the N-terminal tripeptide in the structure of [D-Arg2] dermorphin is of crucial importance for the manifestation of the full intrinsic opioid-like antinociceptive activity of [D-Arg2] dermorphin, which is presumably mediated through opioid receptors in the brain.     

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.     

内吗啡肽及其类似物对心血管系统的作用

目的研究内吗啡肽(EMs)及其类似物对心血管系统的影响，初步探讨其作用机理。方法测定EMs及其类似物对大鼠平均动脉压和后肢血管阻力、蟾蜍肠系膜微动脉内径、兔离体胸主动脉条张力的影响。结果EMs及其类似物剂量依赖(10^-9-10^-6mol·L^-1，iv)且Nx敏感地降低麻醉大鼠平均动脉压、后肢血管灌流压和扩张蟾蜍肠系膜微动脉。EMs对去内皮兔离体胸主动脉条张力无影响；但剂量依赖地显著降低完整内皮胸主动脉条张力并被Nx和L-NNA阻断。结论EMs及其类似物通过降低外周阻力而显著降低动脉血压，其作用与血管内皮细胞释放NO有关。     

Potent and selective antagonists of the antidiuretic responses to arginine-vasopressin based on modifications of [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-D-isoleucine,4- valine]arginine-vasopressin at position 4

As part of a program in which we are attempting (a) to obtain more potent and/or more selective antagonists of the antidiuretic responses to arginine-vasopressin (AVP) and (b) to delineate the structural features at positions 1-9 required for antidiuretic antagonism, we have synthesized 13 new analogues of the antidiuretic antagonist [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-D-isoleucine,4- valine]arginine-vasopressin [d(CH2)5[D-Ile2]VAVP] in which the valine residue at position 4 has been replaced by the L-amino acids Abu, Ile, Thr, Ala, Ser, Nva, Gln, Leu, Lys, Cha, Asn, Orn, and Phe and two new analogues of the antidiuretic antagonist [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-D-phenylalanine,4- valine]arginine-vasopressin [d(CH2)5[D-Phe2]VAVP] with the Val4 residue replaced by Ser and Orn. These analogues are 1, d(CH2)5[D-Ile2,Abu4]AVP; 2, d(CH2)5[D-Ile2,Ile4]AVP; 3, d(CH2)5[D-Ile2,Thr4]AVP; 4, d(CH2)5[D-Ile2,Ala4]AVP; 5, d(CH2)5[D-Ile2,Ser4]AVP; 6, d(CH2)5[D-Ile2,Nva4]AVP; 7, d(CH2)5[D-Ile2]AVP; 8, d(CH2)5[D-Ile2,Leu4]AVP; 9, d(CH2)5[D-Ile2,Lys4]AVP; 10, d(CH2)5[D-Ile2,Cha4]AVP; 11, d(CH2)5[D-Ile2,Asn4]AVP; 12, d(CH2)5[D-Ile2,Orn4]AVP; 13, d(CH2)5[D-Ile2,Phe4]AVP; 14, d(CH2)5[D-Phe2,Ser4]AVP; and 15, d(CH2)5[D-Phe2,Orn4]AVP. The protected peptide precursors for these peptides were prepared by the solid-phase method, followed by ammonolytic cleavage. The free peptides 1-15 were obtained by deblocking with Na in NH3, oxidation of the resultant disulfhydryl compounds with dilute K3[Fe(CN)6], and purification on Sephadex G-15 in a two-step procedure with 50% HOAc and 0.2 M HOAc as eluants. Analogues 1-15 were tested in rats for agonistic and antagonistic activities by antidiuretic, vasopressor, and oxytocic assays.(ABSTRACT TRUNCATED AT 250 WORDS)