title: [D-1-Nal4]endomorphin-2 is a potent micro-opioid receptor antagonist in the aequorin luminescence-based calcium assay.
abstract: A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca(2+) levels, was used to examine relative potency and efficacy of the micro-opioid receptor antagonists. A series of position 3- and 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) analogues containing D-3-(1-naphthyl)-alanine (D-1-Nal) or D-3-(2-naphthyl)-alanine (D-2-Nal), which were previously shown to reverse antinociception induced by endomorphin-2 in the in vivo hot-plate test in mice, was tested in the aequorin luminescence-based calcium assay to examine their micro-opioid antagonist potency in vitro. A recombinant mammalian cell line expressing the micro-opioid receptor together with a luminescent reporter protein, apoaequorin, was used in the study. The results obtained in this functional assay indicated that analogues with D-1-Nal or D-2-Nal substitutions in position 4 of endomorphin-2 are strong micro-opioid receptor antagonists, while those substituted in position 3 are partial agonists. Exceptional antagonist potency in the calcium assay was observed for [D-1-Nal(4)]endomorphin-2. The pA(2) value for this analogue was 7.95, compared to the value of 8.68 obtained for the universal, non-selective opioid antagonist of the alkaloid structure, naloxone. The obtained results were compared with the data from the hot-plate test in mice. In that in vivo assay [D-1-Nal(4)]endomorphin-2 was also the most potent analogue of the series.
authors.affiliation: Laboratory of Biomolecular Chemistry, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland.
authors.forename: Jakub
authors.initials: J
authors.lastname: Fichna
authors.forename: Mariola
authors.initials: M
authors.lastname: Piestrzeniewicz
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Gach
authors.forename: Jeroen
authors.initials: J
authors.lastname: Poels
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Burgeon
authors.forename: Jozef
authors.initials: J
authors.lastname: Vanden Broeck
authors.forename: Anna
authors.initials: A
authors.lastname: Janecka
mesh_terms: D000331:Aequorin / Q000032:analysis; D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D002118:Calcium / Q000032:analysis; D006224:Cricetinae; D004353:Drug Evaluation, Preclinical / Q000379:methods; D008163:Luminescent Measurements; D051379:Mice; D009292:Narcotic Antagonists; D009842:Oligopeptides / Q000737:chemistry* / Q000494:pharmacology*; D011957:Receptors, Opioid / Q000819:agonists
chemical_list: D009292:Narcotic Antagonists; D009842:Oligopeptides; D011957:Receptors, Opioid; C513510:endomorphin-2, 1-Nal(4)-; D000331:Aequorin; D002118:Calcium