A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca2+ levels, was used to examine relative potency and efficacy of the mu-opioid receptor antagonists. A series of position 3- and 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) 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 g-opioid antagonist potency in vitro. A recombinant mammalian cell line expressing the mu-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 R-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. (c) 2006 Elsevier Inc. All rights reserved.