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

https://doi.org/10.1016/0922-4106(93)90005-TGet rights and content

Abstract

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.

References (28)

  • T.J. Haley et al.

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

    Br. J. Pharmacol. Chemother.

    (1957)
  • P.J. Horan et al.

    Agonist and antagonist profiles of [D-Ala2,Glu4]deltorphin and its Cys4- and Ser4-substituted derivatives: further evidence of opioid delta receptor multiplicity

    J. Pharmacol. Exp. Ther.

    (1992)
  • Q. Jiang et al.

    Antinociceptive effects of [D-Ala2]deltorphin II, a highly selective δ agonist in vivo

    Life Sci.

    (1990)
  • Q. Jiang et al.

    Opioid agonist and antagonist antinociceptive properties of [D-Ala2,Leu5,Cys6]-enkephalin selective actions at the deltanon-complexed site

    J. Pharmacol. Exp. Ther.

    (1990)
  • Cited by (0)

    View full text