Pharmacological and functional biochemical properties of D-Ala 2-D-Nle5-enkephalin-Arg-Phe

Fanni Tóth, Gyöngyi Horváth, Margit Szikszay, Judit Farkas, Géza Tóth, Anna Borsodi, Sándor Benyhe

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Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN) a synthetic analogue of the endogenous Met-enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF), was investigated in radioligand binding assays, [35S]GTPγS stimulation experiments as well as in in vivo algesiometric tests. Binding properties of [3H]DADN were measured in crude membrane fractions of rat spinal cord tissues and in homogenates of Chinese hamster ovary (CHO) cells selectively expressing δ-, κ-or μ-opioid receptors. The highest affinity for [3H]DADN binding was observed in membranes from CHO cells transfected with μ-opioid receptors confirming the μ-selectivity of the peptide. Unlabeled DADN was also investigated in functional biochemical experiments by measuring opioid receptor-mediated G-protein activation in rat brain membrane fractions. The peptide stimulated the activity of the regulatory G-proteins in a concentration dependent manner, and the stimulation was efficiently inhibited in the presence of μ-receptor specific antagonist ligands further supporting the selectivity profile of DADN. Intrathecally administered DADN produced a dose-related, naloxone-reversible antinociception in rat hot water tail-flick tests. Among the selective opioid antagonists tested, the δ-selective naltrindole (NTI) and the κ-specific norbinaltorphimine (norBNI) showed only slight blocking effects compared with naloxone. The results obtained in the in vitro agonist-stimulated [ 35S]GTPγS binding assays are in good agreement with the opioid agonist effect seen in the in vivo pain test.

Original languageEnglish
Pages (from-to)139-146
Number of pages8
JournalRegulatory Peptides
Issue number2
Publication statusPublished - Oct 15 2004



  • CHO cells
  • Enkephalins
  • G-protein stimulation
  • Opioid receptor
  • Radioligand binding
  • Tail-withdrawal

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Endocrinology
  • Clinical Biochemistry
  • Cellular and Molecular Neuroscience

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