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

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)25-31
Number of pages7
JournalEuropean Journal of Pharmacology: Molecular Pharmacology
Volume246
Issue number1
DOIs
StatePublished - Jun 15 1993

Fingerprint

Opioid Receptors
D-Penicillamine (2,5)-Enkephalin
Ligands
deltorphin
Sulfhydryl Compounds
Disulfides
Membranes
Enkephalins
Dithiothreitol
Reducing Agents
Brain
Salts

Keywords

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

ASJC Scopus subject areas

  • Pharmacology

Cite this

Pseudoirreversible binding characteristics of [D-Ala2, Glu4]deltorphin and its Cys4 substituted derivative to δ-opioid receptors. / Wild, Kenneth D.; Horan, Peter J.; Misicka, Aleksandra; Lipkowski, Andrzej; Haaseth, Ronald C.; Matsunaga, Terry O.; Hruby, Victor J; Toth, Geza; Borsodi, Anna; Yamamura, Henry I.; Porreca, Frank.

In: European Journal of Pharmacology: Molecular Pharmacology, Vol. 246, No. 1, 15.06.1993, p. 25-31.

Research output: Contribution to journalArticle

Wild, Kenneth D. ; Horan, Peter J. ; Misicka, Aleksandra ; Lipkowski, Andrzej ; Haaseth, Ronald C. ; Matsunaga, Terry O. ; Hruby, Victor J ; Toth, Geza ; Borsodi, Anna ; Yamamura, Henry I. ; Porreca, Frank. / Pseudoirreversible binding characteristics of [D-Ala2, Glu4]deltorphin and its Cys4 substituted derivative to δ-opioid receptors. In: European Journal of Pharmacology: Molecular Pharmacology. 1993 ; Vol. 246, No. 1. pp. 25-31.
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T1 - Pseudoirreversible binding characteristics of [D-Ala2, Glu4]deltorphin and its Cys4 substituted derivative to δ-opioid receptors

AU - Wild, Kenneth D.

AU - Horan, Peter J.

AU - Misicka, Aleksandra

AU - Lipkowski, Andrzej

AU - Haaseth, Ronald C.

AU - Matsunaga, Terry O.

AU - Hruby, Victor J

AU - Toth, Geza

AU - Borsodi, Anna

AU - Yamamura, Henry I.

AU - Porreca, Frank

PY - 1993/6/15

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N2 - 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.

AB - 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.

KW - deltorphin (substituted)

KW - Opioid

KW - Pseudoirreversible binding

KW - δ-Opioid receptor

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