Pseudoirreversible binding characteristics of [D-Ala2, Glu4]deltorphin and its Cys4 substituted derivative to δ-opioid receptors

Kenneth D. Wild, Peter J. Horan, Aleksandra Misicka, Andrzej Lipkowski, Ronald C. Haaseth, Terry O. Matsunaga, Victor J. Hruby, Geza Toth, Anna Borsodi, Henry I. Yamamura, Frank Porreca

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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 Bmax 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.

Original languageEnglish (US)
Pages (from-to)25-31
Number of pages7
JournalEuropean Journal of Pharmacology: Molecular Pharmacology
Issue number1
StatePublished - Jun 15 1993


  • Opioid
  • Pseudoirreversible binding
  • deltorphin (substituted)
  • δ-Opioid receptor

ASJC Scopus subject areas

  • Pharmacology

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