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, A. 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
Pages (from-to)25-31
Number of pages7
JournalEuropean Journal of Pharmacology: Molecular Pharmacology
Volume246
Issue number1
DOIs
Publication statusPublished - 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, A.; 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, KD, Horan, PJ, Misicka, A, Lipkowski, A, Haaseth, RC, Matsunaga, TO, Hruby, VJ, Toth, G, Borsodi, A, Yamamura, HI & Porreca, F 1993, 'Pseudoirreversible binding characteristics of [D-Ala2, Glu4]deltorphin and its Cys4 substituted derivative to δ-opioid receptors', European Journal of Pharmacology: Molecular Pharmacology, vol. 246, no. 1, pp. 25-31. https://doi.org/10.1016/0922-4106(93)90005-T
Wild, Kenneth D. ; Horan, Peter J. ; Misicka, Aleksandra ; Lipkowski, Andrzej ; Haaseth, Ronald C. ; Matsunaga, Terry O. ; Hruby, Victor J. ; Toth, Geza ; Borsodi, A. ; 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, A.

AU - Yamamura, Henry I.

AU - Porreca, Frank

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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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