Pharmacological characterization of [D-Ala2,Leu5,Ser6]enkephalin (DALES): antinociceptive actions at the δnon-complexed-opioid receptor

doi:10.1016/0014-2999(91)90227-H

European Journal of Pharmacology

Volume 192, Issue 3, 17 January 1991, Pages 371-375

https://doi.org/10.1016/0014-2999(91)90227-H Get rights and content

Abstract

Substantial evidence has been accumulated which suggests that opioid δ receptors may be distinguished on the basis of their involvement in the modulation (i.e., increase or decrease in potency) of μ-mediated antinociception. On this basis, it has been hypothesized that some opioid δ receptors exist within a functional complex with μ receptors (δ_complexed (δ_cx) receptors) while other δ sites do not (δ_{non-complexed} (δ_ncx) receptors). Recent work with [D-Ala²,Leu⁵,Cys⁶]enkephalin (DALCE) has demonstrated that this compound produces initial antinociceptive actions, does not modulate morphine antinociception and appears to bind irreversibly to the δ_ncx site, presumably by means of thiol-disulfide exchange between the receptor and the cysteine sulfhydryl group. To determine if a structural basis exists for actions at the hypothesized δ_ncx receptor, in the present study we report the synthesis and pharmacological characterization of [D-Ala²,Leu⁵,Ser⁶]enkephalin (DALES), a close structural analogue of DALCE. If a structural basis for action at the δ_ncx site exists, then DALES would be predicted to produce antinociception, fail to modulate morphine antinociception and, since it lacks the free sulfhydryl group present in DALCE, fail to exhibit irreversible antagonistic actions; these predictions were supported. Additionally, pretreatment with DALCE at − 24 h, but not with DALES, blocked DALES-induced antinociception. These observations in vivo support the concept of a structural basis for activity at the hypothesized δ_ncx site and suggest that DALES, like DALCE, may be a useful probe for pharmacological characterization of putative δ receptor subtypes.

References (24)

R.W. Barrett et al.
The effects of receptor selective opioid peptides on morphine-induced analgesia
European J. Pharmacol.
(1982)
R. Cotton et al.
ICI 174,864: a highly selective antagonist for the opioid δ receptor
European J. Pharmacol.
(1984)
J.S. Heyman et al.
Modulation of μ-mediated antinociception by δ agonists: Characterization with antagonists
European J. Pharmacol.
(1989)
J.S. Heyman et al.
Estimation of the affinity of naloxone at supraspinal and spinal opioid receptors in vivo: studies with receptor selective agonists
Life Sci.
(1986)
J.S. Heyman et al.
Opioid δ receptor involvement in supraspinal and spinal antinociception in mice
Brain Res.
(1987)
J.S. Heyman et al.
Modulation of μ-mediated antinociception by δ agonists in the mouse: Selective potentiation of morphine by [D-Pen²,D-Pen⁵]enkephalin
European J. Pharmacol.
(1989)
J.S. Heyman et al.
Can supraspinal δ-opioid receptors mediate antinociception?
Trends Pharmacol. Sci.
(1988)
N.M. Lee et al.
Opiate and peptide interaction: effect of enkephalins on morphine analgesia
European J. Pharmacol.
(1980)
W.K. Bowen et al.
Affinity labelling of δ-opiate receptors using [D-Ala²,Leu⁵,Cys⁶]enkephalin: Covalent attachment via thio-disulfide exchange
J. Biol. Chem.
(1987)
J.J. Calcagnetti et al.
[D-Ala²,Leu⁵,Cys⁶]enkephalin: Short-term agonist effects and long-term antagonism at δ opioid receptors
Peptides
(1989)

T.J. Haley et al.

Pharmacological effects produced by intracerebral injections of drugs in the conscious mouse

Br. J. Pharmacol. Chemother.

(1957)

J.W. Holaday et al.

Evidence for kapa, μ and δ opioid-binding site interactions in vivo

Cited by (13)

Quantitative autoradiographic mapping of opioid receptors in the brain of δ-opioid receptor gene knockout mice
2002, Brain Research
Citation Excerpt :
It is worthy of note that those regions in heterozygous mice that show rather less than 50% loss of δ-receptors (e.g., habenula, lateral septum and superior colliculus) are also regions which have a high level of μ-receptors and thus a proportion of the binding measured is probably due to μ-cross labeling. There is substantial pharmacological and molecular evidence to suggest heterogeneity of δ-receptors [10,14,18,25,26,28,44]. Only a single gene, Oprd1, has been identified encoding the δ-receptor although the cloned receptor has been reported to exhibit higher affinity for putative δ2-receptor selective ligands than for δ1-selective compounds [31].
Using quantitative receptor autoradiography we have determined if deletion of the δ-opioid receptor gene (Oprd1) results in compensatory changes in the expression of other opioid receptors. Gene targeting was used to delete exon 1 of the mouse δ-opioid receptor gene and autoradiography was carried out on brains from wild-type, heterozygous and homozygous knockout mice. δ-Opioid receptors were labeled with [³H]deltorphin I (7 nM), μ- with [³H]DAMGO (4 nM), and κ- with [³H]CI-977 (2.5 nM) or [³H]bremazocine (2 nM in the presence of DPDPE and DAMGO) and non-specific binding determined with naloxone. [³H]Deltorphin I binding was reduced by approximately 50% in heterozygous animals. In homozygous animals specific binding could only be detected after long-term film exposure (12 weeks). Regions exhibiting this residual [³H]deltorphin I binding correlated significantly with those demonstrating high levels of the μ-receptor and were abolished in the presence of the μ-agonist DAMGO. Autoradiographic mapping showed significant overall reductions in [³H]DAMGO and [³H]CI-977 binding throughout the brain following loss of both copies of the Oprd1 gene. In contrast, overall levels of [³H]bremazocine binding were higher in brains from −/− than +/+ mice. Our findings suggest that residual [³H]deltorphin I binding in the brain of δ-receptor gene knockout mice is the result of cross-reactivity with μ-sites and that there are no δ-receptor subtypes derived from a different gene. Changes in μ- and κ-receptor labeling suggest compensatory changes in these subtypes in response to the absence of the δ-receptor. The differences in [³H]CI-977 and [³H]bremazocine binding indicate these ligands show differential recognition of the κ-receptor.
Novel met-enkephalin analogue: A potent systemic mu agonist antinociceptive agent
1995, Pharmacological Research
A new met-enkephalin analogue (compound 82/205) was evaluated for its opioidergic activity in mice. The compound showed antinociception (warm water tail-flick test), tolerance, cross tolerance to morphine and physical dependence. The time course of antinociptive effect of the compound was comparable to morphine. The antinociceptive ED₅₀ (μmol kg⁻¹ i.p.) values for the compound and morphine base were 5.31 and 7.59, respectively. Its antinociceptive effect was blocked by naloxone, β-FNA (mu antagonist) and naloxonazine (mu₁ antagonist) but not by ICI 174,864 (delta antagonist). Naloxone precipitated withdrawal jumpings were 2.6 times less in compound 82/205 treated mice than the morphine treated group. The new analogue compound 82/205 is a potent mu agonist antinociceptive with a possible weak dependence liability.
Opioid peptide receptor studies. 1. Identification of a novel δ-opioid receptor binding site in rat brain membranes
1995, Peptides
Our laboratory was among the first to propose the existence of δ receptor subtypes: a δ site thought to be associated with a μ-δ-opioid receptor complex termed the δ_cx binding site and δ site not associated with the μ-δ-opioid receptor complex, termed the δ_ncx site. In previous studies, we assayed the δ_cx site with [³H][d-Ala²,d-Leu⁵]enkephalin using rat brain membranes depleted of δ_ncx sites by pretreatment with the site-directed acylating agent, (+)-trans-SUPERFIT. In the present study, we investigated, using (+)-trans-SUPERFIT-pretreated membranes, the possibility of heterogeneity of the δ_cx binding site. Two sites were resolved: the δ_cx−1 site at which μ ligands are potent noncompetitive inhibitors and δ ligands are weak competitive inhibitors, and the δ_cx−2 site has a δ-like ligand-selectivity profile, several experiments distinguished it from the δ_ncx site. Two lines of evidence suggest that the δ_ncx site corresponds to the cloned δ receptor. One, the δ receptor was cloned from the NG108-15 cell line, and this receptor, like the δ_ncx binding site, irreversibly binds SUPERFIT and (+)-trans-SUPERFIT. Secondly, administration of δ-antisense DNA selectively decreases δ_ncx binding. Viewed collectively, the major finding of this study is the discovery of a novel SUPERFIT-insensitive and δ-antisense-insensitive δ_cx−2 binding site.
Identification of a human delta opioid receptor: Cloning and expression
1994, Life Sciences
The δ opioid receptor is an important target for analgesic drug development. This report describes the identification of δ opioid receptor clones from human cDNA libraries and the preparation of a human δ receptor cDNA in the pcDNA3 expression vector for transfection studies. The cDNA encodes a 372 amino acid protein that has 93% amino acid identity to mouse and rat δ receptors. COS-7 cells transfected with this clone express over 1.0 pmole receptor/mg protein when measured by saturation binding with [³H]naltrindole. The δ receptor selective ligands NTB, BNTX, [4′-Cl-Phe⁴]DPDPE and [D-Ala², Glu⁴]deltorphin all have Ki values under 10 nM while the affinities of the μ and κ opioid receptor ligands CTAP and U-69593, respectively, are over 4.0 μM. Agonists show binding to multiple affinity states of the receptor consistent with the presence of G-protein coupled and uncoupled forms of the expressed receptor. The 8-fold higher affinity of NTB relative to BNTX suggests that the human δ receptor is of the δ₂ subtype.
Differential binding of opioid peptides and other drugs to two subtypes of opioid δ<inf>ncx</inf> binding sites in mouse brain: Further evidence for δ receptor heterogeneity
1993, Peptides
Research into the functional role of the opioid δ receptor has intensified with the recent in vivo identification of δ receptor subtypes, termed $δ_{1}$ and $δ_{2}$ , which mediate antinociception in the mouse. A variety of data also support the hypothesis of an opioid receptor complex composed of distinct, yet interacting, μ, δ, and perhaps κ binding sites. This model postulates two classes of δ binding sites: a δ binding site not associated with the opioid receptor complex, termed the $δ_{ncx}$ site, and a δ site associated with the receptor complex, termed the $δ_{cx}$ site. A major purpose of this study was to clarify the relationship between the $δ_{ncx}$ binding sites and the $δ_{1}$ and $δ_{2}$ receptors. Mouse brain membranes were depleted of μ sites by pretreatment with the site-directed acylating agent, BIT, and the $δ_{ncx}$ binding sites were labeled with [³H][d-Ala²,d-Leu⁵]enkephalin. Binding surface analysis readily resolved two binding sites ( $δ_{ncx−1}$ and $δ_{ncx−2}$ ) in the absence and presence of 100 mM NaCl. Control experiments with guanine nucleotides and the ligand-selectivity analysis indicated that the two sites were not two states of a single receptor. Pretreatment of membranes with DALCE, but not [Cys⁴]deltorphin, decreased [³H][d-Ala²,d-Leu⁵]enkephalin and [³H][d-Ser²,Thr⁶]enkephalin binding. Ligand-selectivity analysis of the two binding sites suggested that neither $δ_{ncx}$ binding site had the characteristics expected of the $δ_{2}$ receptor, and that the $δ_{ncx−1}$ site, but not the $δ_{ncx−2}$ site, was synonymous with the $δ_{1}$ receptor. Moreover, our finding that the racemic nonpeptide δ agonist, BW373U86, had high affinity at and selectivity for the $δ_{ncx−2}$ site suggests that this site may be a novel δ receptor that mediates some of the effects of BW373U86.
Pseudoirreversible binding characteristics of [D-Ala2, Glu4]deltorphin and its Cys4 substituted derivative to δ-opioid receptors
1993, European Journal of Pharmacology: Molecular Pharmacology
Following the identification of [D-Ala²,Glu⁴]deltorphin as a selective δ₂-opioid receptor agonist in vivo, we synthesized the Cys⁴-substituted analogue as a potential ligand which might bind ‘irreversibly’ at this site through a proposed thil-disulfide exchange mechanism. Previous studies showed that intracerebroventricular (i.c.v.) pretreatment with [D-Ala²,Cys⁴]deltorphin, 24 h prior to antinociceptive testing, produced a selective antagonism of [D-Ala²,Glu⁴]deltorphin-induced antinociception in mice. Surprisingly, however, the Ser⁴-analogue (synthesized as a control) and even the parent molecule, [D-Ala²,Glu⁴]deltorphin, had the same antagonistic effect following pretreatment in vivo, while pretreatment with an equiantinociceptive dose of [D-Ser²,Leu⁵,Thr⁶]-enkephalin, a structurally unrelated δ₂-opioid receptor agonist did not exhibit long-lasting antinociceptive actions. These data raised questions regarding the mechanism of the antagonism observed in vivo with the deltorphins; the present studies have attempted to explore these issues using radioligand binding techniques. The results demonstrate a decrease in the B_max of [tyrosyl-3′,5′-³H,D-Pen²,p-Cl-Phe⁴,D-Pen⁵]-enkephalin ([³H]p-Cl-DPDPE) (δ-opioid receptor ligand) following i.c.v. pretreatment of mice (at −24 h) with [D-Ala²,Cys⁴]deltorphin or [D-Ala²,Glu⁴]deltorphin, but not with [D-Ala²,Ser⁴] deltorphin, suggesting a difference in mechanism of antagonism seen in vivo with these compounds. Incubation of mouse whole brain homogenates in vitro with [D-Ala²,Cys⁴]deltorphin or with [D-Ala²,Glu⁴]deltorphin, also resulted in a decrease in the radioligand binding of [³H]p-Cl-DPDPE, but this effect was not prevented by coincubation with dithiothreitol, a thiol-reducing agent. Direct evaluation of binding using [³H][D-Ala²,Glu⁴]deltorphin (5 nM) showed that a portion of this ligand (i.e., about 10% of all specific binding) remained specifically and ‘irreversibly’ bound to mouse brain membranes following incubation in vitro and extensive washing. The ‘irreversibly’, specifically bound [³H][D-Ala²,Glu⁴]deltorphin could be removed, however, by brief exposure of the membranes to a low pH (2.5), high-salt (0.5 M NaCl) solution. These data suggest that [D-Ala²,Cys⁴] deltorphin and [D-Ala²,Glu⁴]deltorphin bind in a ‘pseudoirreversible’ (no-covalent) manner to an δ-opioid receptor via a mechanism that apparently does not involve thiol-disulfide exchange.

View all citing articles on Scopus

View full text

Article preview

European Journal of Pharmacology

Abstract

References (24)

The effects of receptor selective opioid peptides on morphine-induced analgesia

European J. Pharmacol.

ICI 174,864: a highly selective antagonist for the opioid δ receptor

European J. Pharmacol.

Modulation of μ-mediated antinociception by δ agonists: Characterization with antagonists

European J. Pharmacol.

Estimation of the affinity of naloxone at supraspinal and spinal opioid receptors in vivo: studies with receptor selective agonists

Life Sci.

Opioid δ receptor involvement in supraspinal and spinal antinociception in mice

Brain Res.

Modulation of μ-mediated antinociception by δ agonists in the mouse: Selective potentiation of morphine by [D-Pen²,D-Pen⁵]enkephalin

European J. Pharmacol.

Can supraspinal δ-opioid receptors mediate antinociception?

Trends Pharmacol. Sci.

Opiate and peptide interaction: effect of enkephalins on morphine analgesia

European J. Pharmacol.

Affinity labelling of δ-opiate receptors using [D-Ala²,Leu⁵,Cys⁶]enkephalin: Covalent attachment via thio-disulfide exchange

J. Biol. Chem.

[D-Ala²,Leu⁵,Cys⁶]enkephalin: Short-term agonist effects and long-term antagonism at δ opioid receptors

Peptides

Pharmacological effects produced by intracerebral injections of drugs in the conscious mouse

Br. J. Pharmacol. Chemother.

Evidence for kapa, μ and δ opioid-binding site interactions in vivo

Cited by (13)

Quantitative autoradiographic mapping of opioid receptors in the brain of δ-opioid receptor gene knockout mice

Novel met-enkephalin analogue: A potent systemic mu agonist antinociceptive agent

Opioid peptide receptor studies. 1. Identification of a novel δ-opioid receptor binding site in rat brain membranes

Identification of a human delta opioid receptor: Cloning and expression

Differential binding of opioid peptides and other drugs to two subtypes of opioid δ<inf>ncx</inf> binding sites in mouse brain: Further evidence for δ receptor heterogeneity

Pseudoirreversible binding characteristics of [D-Ala<sup>2</sup>, Glu<sup>4</sup>]deltorphin and its Cys<sup>4</sup> substituted derivative to δ-opioid receptors

Pharmacological characterization of [D-Ala2,Leu5,Ser6]enkephalin (DALES): antinociceptive actions at the δnon-complexed-opioid receptor

Abstract

European J. Pharmacol.

European J. Pharmacol.

European J. Pharmacol.

Life Sci.

Brain Res.

European J. Pharmacol.

Trends Pharmacol. Sci.

European J. Pharmacol.

Affinity labelling of δ-opiate receptors using [D-Ala2,Leu5,Cys6]enkephalin: Covalent attachment via thio-disulfide exchange

J. Biol. Chem.

[D-Ala2,Leu5,Cys6]enkephalin: Short-term agonist effects and long-term antagonism at δ opioid receptors

Peptides

Pharmacological effects produced by intracerebral injections of drugs in the conscious mouse

Br. J. Pharmacol. Chemother.

Evidence for kapa, μ and δ opioid-binding site interactions in vivo

Pharmacological characterization of [D-Ala²,Leu⁵,Ser⁶]enkephalin (DALES): antinociceptive actions at the δ_{non-complexed}-opioid receptor

Affinity labelling of δ-opiate receptors using [D-Ala²,Leu⁵,Cys⁶]enkephalin: Covalent attachment via thio-disulfide exchange

[D-Ala²,Leu⁵,Cys⁶]enkephalin: Short-term agonist effects and long-term antagonism at δ opioid receptors