A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist,...

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [(35)S]GTPγS functional assays were performed in the presence of putative δ(1)-(DPDPE: agonist, BNTX: antagonist), δ(2)-(agonist: deltorphin II, Ile(5,6)-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ(2)- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with K(D)=0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [(3)H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [(3)H]Ile(5,6)-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49±0.06 and 0.30±0.01 nM potency against DPDPE and deltorphin II in the [(35)S]GTPγS functional assay, respectively. The rank order of potency of putative δ(1)- and δ(2)-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ(1)- and δ(2)-opioid receptors could not be unequivocally distinguished in vitro.