Highly potent side chain–main chain cyclized dermorphin–deltorphin analogues: An integrated approach including synthesis,bioassays, NMR spectroscopy and molecular modelling

Our continuing efforts to study structure–activity relationships of peptide opioids have resulted in the synthesis of a series of cyclic opioids related to dermorphins and deltorphins. The biological activities of the compounds have been determined and the conformational analyses carried out using ¹H-NMR spectroscopy and molecular modelling. The three compounds in the series Tyr-c[D -Orn-Phe-Ala], Tyr-c[D -Lys-Phe-Ala], and Tyr-c[A₂Bu-Phe-Ala-Leu] are cyclized via a lactam bridge from the side-chain of the residue at the second position with the carboxyl terminus of each compound. The molecules incorporate 12-, 13- and 14-membered rings, respectively. They include a phenylalanine at the third position which is a distinguishing characteristic of dermorphins and deltorphins. The guinea pig ileum and mouse vas deferens assays show that the compounds are highly active at both μ- and δ-opioid receptors. The compounds are all highly effective antinociceptive agents as measured by the intrathecal rat hot plate test. Conformational analyses of the molecules indicate that they can adopt topochemical arrays required for bioactivity at both μ- and δ-receptors which explains their high activity in both guinea pig ileum and mouse vas deferens in vitro assays. The results support our models for μ- and δ-receptor activity for constrained peptide opioids.     

Design,synthesis and SAR analysis of novel selective σ1 ligands (Part 2)

In order to investigate the molecular features involved in sigma receptors (σ-Rs) binding, new compounds based on arylalkylaminoalcoholic, arylalkenyl- and arylalkylaminic scaffolds were synthesized and their affinity towards σ₁- and σ₂-Rs subtypes was evaluated. The most promising compounds were also screened for their affinity at μ-opioid, δ-opioid and κ-opioid receptors. Biological results are herein presented and discussed.     

Glucagon-like peptide-1(1-37) can enhance blood glucose homeostasis in mice

Tyrosyl O-sulfation is a common posttranslational derivatization of proteins that may also modify regulatory peptides. Among these are members of the cholecystokinin (CCK)/gastrin family. While sulfation of gastrin peptides is without effect on the bioactivity, O-sulfation is crucial for the cholecystokinetic activity (i.e. gallbladder emptying) of CCK peptides. Accordingly, the purification of CCK as a sulfated peptide was originally monitored by its gallbladder emptying effect. Since then, the dogma has prevailed that CCK peptides are always sulfated. The dogma is correct in a semantic context since the gallbladder expresses only the CCK-A receptor that requires sulfation of the ligand. CCK peptides, however, are also ligands for the CCK-B receptors that do not require ligand sulfation. Consequently, unsulfated CCK peptides may act via CCK-B receptors. Since in vivo occurrence of unsulfated products of proCCK with an intact α-amidated C-terminal tetrapeptide sequence (-Trp-Met-Asp-PheNH(2)) has been reported, it is likely that unsulfated CCK peptides constitute a separate hormone system that acts via CCK-B receptors. This review discusses the occurrence, molecular forms, and possible physiological as well as pathophysiological significance of unsulfated CCK peptides.     

Activation of CCK-A receptors induces elevation of plasma corticosterone in rats.

Cholecystokinin octapeptide (CCK-8) and ceruletide (1 microgram/kg) produced a pronounced increment of plasma corticosterone levels at 30 min after intraperitoneal administration. The response to these peptides was suppressed by pretreatment with a selective antagonist for CCK-A receptors, (-)L-364,718, in a dose-related manner, but not with an antagonist for CCK-B receptors, (+)L-365,260. However, (-)L-364,718 itself had no effect on basal levels of plasma corticosterone. These results indicate that peripheral administration of CCK-8 and ceruletide stimulates the hypothalamo-pituitary adrenal axis through the activation of CCK-A receptors, but not CCK-B receptors.     

Cyclic side-chain-linked opioid analogs utilizing cis- and trans-4-aminocyclohexyl-d-alanine

Cyclization of linear sequences is a well recognized tool in opioid peptide chemistry for generating analogs with improved bioactivities. Cyclization can be achieved through various bridging bonds between peptide ends or side-chains. In our earlier paper we have reported the synthesis and biological activity of a cyclic peptide, Tyr-c[d-Lys-Phe-Phe-Asp]NH₂ (1), which can be viewed as an analog of endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH₂). Cyclization was achieved through an amide bond between side-chains of d-Lys and Asp residues. Here, to increase rigidity of the cyclic structure, we replaced d-Lys with cis- or trans-4-aminocyclohexyl-d-alanine (d-ACAla). Two sets of analogs incorporating either Tyr or Dmt (2′,6′-dimethyltyrosine) residues in position 1 were synthesized. In the binding studies the analog incorporating Dmt and trans-d-ACAla showed high affinity for both, μ- and δ-opioid receptors (MOR and DOR, respectively) and moderate affinity for the κ-opioid receptor (KOR), while analog with Dmt and cis-d-ACAla was exceptionally MOR-selective. Conformational analyses by NMR and molecular docking studies have been performed to investigate the molecular structural features responsible for the noteworthy MOR selectivity.     

Characterization of the bioactive form of linear peptide antagonists at the δ-opioid receptor

The stereochemical requirements for δ-opioid receptor binding of a series of linear peptide antagonists with a novel conformationally restricted Phe analogue (Tic) as a second residue were examined by using a variety of computational chemistry methods. The δ-opioid receptor analogues with significant affinity, Tyr-Tic-NH₂ (TI-NH₂), Tyr-Tic-Phe-OH (TIP), Tyr-Tic-Phe-NH₂(TIP-NH₂), Tyr-Tic-Phe-Phe-OH (TIPP), Tyr-Tic-Phe-Phe-NH₂) (TIPP-NH₂), and the low affinity δ-opioid peptides Tyr-Pro-Phe-Pro-NH₂ (morphiceptin) and Tyr-Phe-Phe-Phe-NH₂ (TPPP-NH₂), were included in this study. The conformational profiles of these peptides were obtained by consecutive cycles of high and low temperature molecular dynamic simulations, coupled to molecular mechanical energy minimization carried out until no new conformational minima were obtained. Comparing the results for TPPP-NH₂ and TIPP-NH₂, the presence of the conformationally restricted Tic residue did not greatly reduce the number of unique low energy conformations, but did allow low energy conformers involving cis bonds between the first two residues. The conformational libraries of these peptides were examined for their ability to satisfy the three key ligand components for receptor recognition already identified by previous studies of high affinity cyclic (Tyr¹-D -Pen²-Gly³-Phe⁴-D -Pen⁵) enkephalin (DPDPE) type agonists: a protonated amine group, an aromatic ring, and a lipophilic moiety in a specific geometric arrangement. Two types of conformations common to the five high δ-opioid affinity L -Tic analogues were found that satisfied these requirements, one with a cis and the other with a trans peptide bond between the Tyr¹ and Tic² residues. Moreover, both the Tic² and Phe³ residues could mimic the hydrophobic interactions with the receptor of the Phe⁴ moiety in the cyclic DPDPE type agonists, consistent with the appreciable affinity of both di-and tripeptides. The low δ-opioid receptor affinity of morphiceptin can be understood as the result of conformational preferences that prevent the fulfillment of this pharmacophore for recognition. © 1996 John Wiley & Sons, Inc.     

Functional CCK-A and Y2 receptors in guinea pig esophagus

Effects of cholecystokinin octapeptide (CCK-8), peptide YY (PPY), neuropeptide Y (NPY) and their analogs on muscle contractions of esophageal strips were investigated. CCK-8 induced a tetrodotoxin and atropine-sensitive contraction. The relative potencies for CCK related peptides to induce contractions were CCK-8 > desulfated CCK-8 > gastrin-17-I. The CCK-A receptor antagonist L-364,718 was 300-fold more potent than the CCK-B receptor antagonist L-365,260 at inhibiting CCK-8-induced contraction. These indicate that neural CCK-A receptors mediate this contraction. PYY or NPY did not cause muscle contraction or inhibit muscle contraction induced by carbachol, endothelin-1 or KCl. However, both PYY and NPY concentration-dependently inhibited contraction induced by CCK-8. This inhibition was not affected by nitric oxide (NO) synthase inhibitors L-NMMA or L-NAME. The relative potencies of PYY related peptides to inhibit CCK-8 induced contraction were PYY > NPY > NPY13-36 > [Leu(31), Pro(34)]NPY > pancreatic polypeptide (PP). We conclude that CCK interacts with neural CCK-A receptors to cause esophageal muscle contraction. PYY and NPY interact with Y2 receptors to inhibit this CCK-induced muscle contraction by an effect not related to NO.     

Differential Regulation of D1 Dopamine Receptor- and of A2a Adenosine Receptor-Stimulated Adenylyl Cyclase by μ-, δ1-, and δ2-Opioid Agonists in Rat Caudate Putamen

Abstract: Inhibition and stimulation of adenylyl cyclase by opioid and D₁ dopamine or A_2a adenosine agonists, respectively, were characterized in the caudate putamen of rats. D₁ dopamine receptors have been reported to be localized preferentially on striatonigral neurons and A_2a adenosine receptors on striatopallidal neurons. The aim of the present study was to evaluate the effects of μ-[Tyr-d -Ala-Gly-(N-Me)Phe-Gly-ol (DAMGO)], δ₁-[Tyr-d -Pen-Gly-Phe-d -Pen (DPDPE)], and δ₂- ([d -Ala²]deltorphin-II [DT-II]) opioid agonists on the D₁ dopamine receptor- and A_2a adenosine receptor-stimulated adenylyl cyclase in membranes from rat caudate putamen. The results show that DAMGO, DPDPE, and DT-II inhibit forskolin-stimulated adenylyl cyclase [selectively antagonized by d -Phe-Cys-Tyr-d -Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP; μ antagonist), 7-benzylidenenaltrexone (BNTX; δ₁ antagonist), and naltriben (NTB; δ₂ antagonist), respectively], but only μ- and δ₂-opioid agonists inhibit D₁ dopamine-stimulated adenylyl cyclase (antagonized by CTOP and NTB, respectively). Furthermore, DT-II and DPDPE inhibit A_2a adenosine-stimulated adenylyl cyclase (antagonized by NTB and BNTX, respectively), whereas DAMGO did not inhibit A_2a adenosine-stimulated adenylyl cyclase activity. These results suggest that μ-, δ₁-, and δ₂-opioid receptors display differential localization and provide neurochemical evidence suggesting the differential location of the δ₁ and δ₂ subtypes. μ-Opioid receptors may be preferentially expressed by striatonigral neurons, δ₁- by striatopallidal neurons, and δ₂- by these two striatal efferent neuron populations.     

Development of a model for the δ-opioid receptor pharmacophore: 3. Comparison of the cyclic tetrapeptide Tyr-c[D-Cys-Phe-D-Pen] OH with other conformationally constrained δ-receptor selective ligands

We have previously proposed a model of the δ-opioid receptor bound conformation for the cyclic tetrapeptide, Tyr-c[D -Cys-Phe-D -Pen]OH (JOM-13) based on its conformational analysis and from conformation-affinity relationships observed for its analogues with modified first and third residues. To further verify the model, it is compared here with results of conformational and structure-activity studies for other known conformationally constrained δ-selective ligands: the cyclic pentapeptide agonist, Tyr-c[D -Pen-Gly-Phe-D -Phe]OH (DPDPE); the peptide antagonist, Tyr-Tic-Phe-PheOH (TIPP); the alkaloid agonist, 7-spiroindanyloxymorphone (SIOM); and the related alkaloid antagonist, oxymorphindole (OMI). A candidate δ-bound conformer is identified for DPDPE that provides spatial overlap of the functionally important N-terminal N⁺₃ and C-terminal COO⁻ groups and the aromatic rings of the Tyr and Phe residues in both cyclic peptides. It is shown that all δ-selective ligands considered have similar arrangements of their pharmacophoric elements, i.e., the tyramine moiety and a second aromatic ring (i.e., the rings of Phe³, Phe⁴, and Tic² residues in JOM-13, DPDPE, and TIPP, respectively; the indole ring system in OMI, and the indanyl ring system in SIOM). The second aromatic rings, while occupying similar regions of space throughout the analogues considered, have different orientations in agonists and antagonists, but identical orientations in peptide and alkaloid ligands with the same agonistic or antagonistic properties. These results agree with the previously proposed binding model for JOM-13, are consistent with the view that δ-opioid agonists and antagonists share the same binding site, and support the hypothesis of a similar mode of binding for opioid peptides and alkaloids. © 1996 John Wiley & Sons, Inc.     

The role of δ-opioid receptor subtypes in cocaine- and methamphetamine-induced place preferences

The effects of δ-receptor antagonists on cocaine- and methamphetamine-induced place preferences were examined in rats. Cocaine- and methamphetamine-induced place preferences were significantly attenuated by naltrindole (NTI: a non-selective δ-opioid receptor antagonist). Furthermore, naltriben (NTB: a selective δ₂-opioid receptor antagonist), but not 7-benzylidenenaltrexone (BNTX: a selective δ₁-opioid receptor antagonist), attenuated the cocaine- and methamphetamine-induce place preferences. These results suggest that δ-opioid receptors, particularly δ₂-opioid receptors, may be involved in the reinforcing effects of cocaine and methamphetamine.     

Inhibition of goby posterior intestinal NaCl absorption by natriuretic peptides and by cardiac extracts

Natriuretic peptides abolish active Na⁺ and Cl^- absorption aross the posterior intestine of the euryhaline gobyGillichthys mirabilis. Inhibition by eel and human natriuretic peptides is dose-dependent with the following sequence of potencies based on experimentally determined ID₅₀ values for inhibition of short-circuit current: eel ventricular natriuretic peptide (78 nmol · l^-1), eel atrial natriuretic peptide (156 nmol · l^-1), human brain natriuretic peptide (326 nmol · l^-1), human α atrial natriuretic peptide (1.05 μmol · l^-1), and eel C-type natriuretic peptide (75 μmol · l^-1). Natriuretic peptides also significantly increase transcellular conductance. The observed sequence of natriuretic peptide potencies is suggestive of cellular mediation by GC-A-type NP-R₁ receptors in this tissue; as expected for guanylyl-cyclase-coupled NP-R₁ receptors, cyclic GMP mimics the action of natriuretic peptides on the goby intestine. Crude aqueous extracts of goby atrium and ventricle inhibited short circuit current and increased tissue conductance in a dose-dependent manner. Ventricular extract was more potent than atrial extract on both a per organ and per milligram basis.     

Buprenorphine exerts its antinocicepttve activity via μ1-opioid receptors

The mechanism of the antinociceptive effect of buprenorphine was assessed by administering selective μ-, μ₁-, δ- and κ-opioid receptor antagonists in mice. Intraperitoneal administration of buprenorphine, at doses of 0.3 to 3 mg/kg, produced dose-dependent antinociception in the tail-flick test. The antinociceptive activity of buprenorphine did not result from the activation of κ- or δ-opioid receptors, since treatment with either nor-binaltorphimine, a selective κ-opioid receptor antagonist, or naltrindole, a selective δ-opioid receptor antagonist, was completely ineffective in blocking buprenorphine-induced antinociception. However, the antinociceptive effect of buprenorphine was significantly antagonized by β-funaltrexamine, a selective μ-opioid receptor antagonist. Moreover, selective μ₁-opioid receptor antagonists, naloxonazine and naltrexonazine, also significantly antagonized the antinociceptive effect of buprenorphine. Co-administration of κ- and δ-opioid receptor antagonists with the μ-opioid receptor antagonists had no significant effect on the antagonistic profiles of the μ-opioid receptor antagonists on the antinociceptive effect of buprenorphine. These results suggest that buprenorphine acts selectively at μ₁-opioid receptors to induce antinociceptive effects in mice.     

Development of new potent agonists able to interact with two postulated subsites of the cholecystokinin CCK-B receptor

Summary Since the biochemical and pharmacological profile of BC 197 and BC 264, two CCK₈-derived agonists with high specificity for CCK-B receptors, suggests their potential interaction with two CCK-B receptor subsites, it appeared essential to design new series of compounds that would be able to discriminate between these two subsites. As CCK₄ is the shortest fragment of CCK which interacts selectively with CCK-B receptors, compounds derived from the C-terminal tetrapeptide domain of BC 264, Boc-Trp-(NMe)Nle-Asp-Phe-NH₂, and of the cyclic compound BC 197, were prepared. While RB 360 (N-(cycloamido)-α-Me(R)Trp-[(2S)-2-amino-9-((cycloamido)carbonyl)nonanoyl]-Asp-Phe-NH₂), like BC 197, has a CCK-B₁ profile with anxiogenic-like effects in the elevated plus-maze test, RB 400 (HOOC-CH₂-CO-Trp-(NMe)Nle-Asp-Phe-NH₂), like BC 264, seems to be a specific CCK-B₂ agonist, able to increase attention and/or memory processes in the Y-maze test.     

Replacement of Tyr-SO3H by a p-carboxymethyl-phenylalanine in a CCK8-derivative preserves its high affinity for CCK-B receptor.

The sulfated tyrosine present in the sequence of CCK8 Asp26-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-PheNH2, seems to play a critical role in the recognition of CCK-A binding sites. In this work, we have investigated whether the presence of an anionic charge on the tyrosine moiety is strictly necessary and whether the sulfate moiety interacts with a divalent cation in the receptor subsite. For this purpose, the novel amino acids (L,D)Phe(p-CH2CO2H) and (L,D) Phe(p-CH2CONHOH), as well as their L-resolved forms were introduced into the sequence of Ac[X27, Nle28, Nle31]-CCK27-33 by solid phase method. The biological activities of these new derivatives were compared to two almost equiactive analogues of CCK8, Ac[Phe(p-CH2SO3H)27, Nle28, Nle31]-CCK27-33 and Boc[Nle28, Nle31]-CCK27-33 (BDNL) and to the nonsulfated analogue of the latter peptide (BDNL NS). All these new CCK-related analogues behave as agonists in stimulating pancreatic amylase release and display high affinity for brain binding sites (KI approximately 3-11 nM) but the only peptides which retain affinity for CCK-A receptors (KI approximately 20 nM) are those containing a p-carboxymethyl phenylalanine. Thus, introduction of this amino acid under an esterified form on the side chain, into specific and potent CCK-B agonists could allow compounds endowed with good bioavailabilities to be obtained.     

A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [³⁵S]GTPγS functional assays were performed in the presence of putative δ₁-(DPDPE: agonist, BNTX: antagonist), δ₂-(agonist: deltorphin II, Ile^5,6-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ₂- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with K_D = 0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [³H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [³H]Ile^5,6-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49 ± 0.06 and 0.30 ± 0.01 nM potency against DPDPE and deltorphin II in the [³⁵S]GTPγS functional assay, respectively. The rank order of potency of putative δ₁- and δ₂-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ₁- and δ₂-opioid receptors could not be unequivocally distinguished in vitro.     

The μ-selective Heptapeptide Opioid Dermorphin has Two Conformations Around Phe3 Ψ with no Head-to-tail Interaction. A Quantitative 2-D NMR and Molecular Modeling Analysis

Abstract

The μ opioid heptapeptide Dermorphin (DRM) is under 70% of trans forms for the Tyr⁵-Pro⁶ peptide bond in solution (CDC1₃/DMSO-d₆ 1/1 vol/vol). Variations of NOE integrals at 5 temperatures show apparent correlation times of 0.8 to 0.9 ns (at 280 K) in that mixed solvent. Four NOE between non-adjacent residues reveal a large population of folded structures. However, in trans DRM, 4 adjacent NOE Phe³/Gly⁴ can only be explained by an equilibrium between folded (ψ³ < 0) and extended (ψ³ > 0) conformations. Simulated annealing modeling gave about 60% (ψ³ < 0) and 40% (ψ³ > 0) of these conformer populations.

Trans DRM study and previous studies on the heptapeptide opioids, dermenkephalin (DREK) and deltorphin-I (δ selective), and DREK(1–4)-DRM(5–7) hybrid (μ selective), show in folded structures more backbone bending of the first 4 residues in the μ opioids than in the δ peptides. Also, the main difference between μ- and δ-opioid peptides is a large fraction of extended conformations in μ heptapeptides. Either bending of the N-terminus, or extension of the C-terminal part in μ-opioid heptapeptides prevent the head-to-tail interactions which allow δ-opioid peptides to bind selectively to the δ-opioid receptor.     

Quantitative structure-activity relationship study on some nonpeptidal cholecystokinin antagonists.

A quantitative structure-activity relationship (QSAR) analysis has been performed on a series of 1,4-benzodiazepine derivatives, which were found to act as antagonists of cholecystokinin (CCK), a gastrointestinal peptide hormone. The CCK acts with three different receptor subtypes termed as CCK-A, CCK-B, and gastrin receptor, which can be found in peripheral system, brain, and stomach, respectively. With all the three subtypes, the binding of the compounds is found to significantly depend on the lipophilicity of the compounds and their ability to form the hydrogen bonds with the receptor. However, the binding sites in CCK-A receptor seem to be slightly rigid as compared to those in CCK-B or gastrin receptor. The latter two appear to have similar binding features.     

Effects of C-Terminal Truncation of the Recombinant δ-Opioid Receptor on Phospholipase C and Adenylyl Cyclase Coupling

Abstract: Opioid receptors belong to the superfamily of guanine nucleotide binding (G) protein-coupled receptors. There is now growing evidence in support of a stimulatory coupling of opioid receptors to phospholipase C (PLC), via a pertussis toxin-sensitive G protein, leading to the generation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]. We have generated two C-terminal truncation mutants of the δ-opioid receptor lacking the final 15 or 37 amino acids and examined their coupling to PLC and adenylyl cyclase. d -[Pen^2,5]-enkephalin (DPDPE) mediated Ins(1,4,5)P₃ formation and cyclic AMP inhibition was measured in whole cells and assayed using radioreceptor mass assays. DPDPE produced a time- and dose-dependent increase in Ins(1,4,5)P₃ mass formation in Chinese hamster ovary (CHO) cells expressing the δ_wt, δ₁₅, and δ₃₇ receptors. As the C terminus was truncated, the time to maximum stimulation (15 s in CHOδ_wt, 60 s in CHOδ₁₅, and 120 s in CHOδ₃₇) increased and removal of the C terminus resulted in a prompt return to basal Ins(1,4,5)P₃ levels. Whereas the dose-response curves to Ins(1,4,5)P₃ formation and cyclic AMP inhibition remained largely unaffected by C-terminal truncation, there were large differences in the pEC/IC₅₀ values, with cyclic AMP inhibition being the more potent, perhaps indicating G_iα coupling to adenylyl cyclase and G_iβ/γ coupling to PLC. Collectively, these data indicate that the C terminus of the δ-opioid receptor is unimportant in the acute coupling to adenylyl cyclase but may have a role to play in PLC coupling. We hypothesize that an intact C terminus is required to allow normal “strong” coupling of receptor to G_i and that truncation weakens this link as reflected in an increased time to peak. In addition, if the coupling is weak, the acute response to agonist stimulation rapidly uncouples.     

Streptozotocin-induced diabetes selectively enhances antinociception mediated by δ1-but not δ2-opioid receptors

We assessed the effect of diabetes on antinociception produced by intracerebroventricular injection of δ-opioid receptor agonists [D-Pen^2,5]enkephalin (DPDPE) and [D-Ala²]deltorphin II. The antinociceptive effect of DPDPE (10 nmol), administered i.c.v., was significantly greater in diabetic mice than in non-diabetic mice. The antinociceptive effect of i.c.v. DPDPE was significantly reduced in both diabetic and non-diabetic mice following pretreatment with 7-benzylidenenaltrexone (BNTX), a selective δ₁-opioid receptor antagonist, but not with naltriben (NTB), a selective δ₂- opioid receptor antagonist. There were no significant differences in the anticiceptive effect of [D-Ala²]deltorphin II (3 nmol, i.c.v.) in diabetic and non-diabetic mice. Furthermore, the antinociceptive effect of i.c.v. [D-Ala²]deltorphin II was significantly reduced in both diabetic and non-diabetic mice following pretreatment with NTB, but not with BNTX. In conclusion, mice with diabetes are selectively hyper-responsive to supraspinal δ₁-opioid receptor-mediated antinociception, but are normally responsive to activation of δ₂-opiod receptors.     

Involvement of spinal release of α-neo-endorphin on the antinociceptive effect of TAPA

The antinociceptive effect of i.t.-administered Tyr-d-Arg-Phe-β-Ala (TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the mouse tail-flick test, TAPA administered i.t. produced a potent antinociception. The antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with the κ-opioid receptor antagonist nor-binaltorphimine, as well as by the μ-opioid receptor antagonist β-funaltrexamine and the μ₁-opioid receptor antagonist naloxonazine. TAPA-induced antinociception was also significantly suppressed by co-administration of the μ₁-opioid receptor antagonist Tyr-d-Pro-Phe-Phe-NH₂ (d-Pro²-endomorphin-2) but not by co-administration of the μ₂-opioid receptor antagonists Tyr-d-Pro-Trp-Phe-NH₂ (d-Pro²-endomorphin-1) and Tyr-d-Pro-Trp-Gly-NH₂ (d-Pro²-Tyr-W-MIF-1). In CXBK mice whose μ₁-opioid receptors were naturally reduced, the antinociceptive effect of TAPA was markedly suppressed compared to the parental strain C57BL/6ByJ mice. Moreover, the antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with antiserum against the endogenous κ-opioid peptide α-neo-endorphin but not antisera against other endogenous opioid peptides. In prodynorphin-deficient mice, the antinociceptive effect of TAPA was significantly reduced compared to wild-type mice. These results suggest that the spinal antinociception induced by TAPA is mediated in part through the release of α-neo-endorphin in the spinal cord via activation of spinal μ₁-opioid receptors.