The ability of several p-selective opioid peptides to activate G-proteins was measured in rat thalamus membrane preparations. The mu-selective ligands used in this study were three structurally related peptides, endomorphin-1, endomorphin-2 and morphiceptin, and their analogs modified in position 3 or 4 by introducing 3-(1-naphthyl)-D-alanine (D-1-Nal) or 3-(2-naphthyl)-D-alanine (D-2-Nal). The results obtained for these peptides in [S-35]GTP gamma S binding assay were compared with those obtained for a standard mu-opioid agonist DAMGO. [D-1-Nal(3)]Morphiceptin was more potent in G-protein activation (EC50 value of 82.5 +/- 4.5 nM) than DAMGO (EC50 = 105 +/- 9 nM). [D-2-Nal(3)]Morphiceptin, as well as endomorphin-2 analogs substituted in position 4 by either D-1-Nal or D-2-Nal failed to stimulate [S-35]GTP gamma S binding and were shown to be potent antagonists against DAMGO. It seems that the topographical location of the aromatic ring of position 3 and 4 amino acid residues can result in a completely different mode of action, producing either agonists or antagonists. (c) 2006 Elsevier Inc. All rights reserved.