A comprehensive study on the putative x3b4;-opioid receptor (sub)types using the highly selective x3b4;-antagonist, Tyr-Tic-(2S,3R)-x3b2;-MePhe-Phe-OH
文摘
The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-x3b2;-MePhe-Phe-OH and see if putative x3b4;-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 [35S]GTPx3b3;S functional assays were performed in the presence of putative x3b4;1-(DPDPE: agonist, BNTX: antagonist), x3b4;2-(agonist: deltorphin II, Ile5,6-deltorphin II, antagonist: naltriben) and x3bc;-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60 % , but did not antagonize x3b4;2- and x3bc;-agonist induced analgesia. The radiolabeled form identified binding sites with KD = 0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [3H]Tyr-Tic-(2S,3R)-x3b2;-MePhe-Phe-OH agreed well with that of [3H]Ile5,6-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-x3b2;-MePhe-Phe-OH displayed 2.49 ± 0.06 and 0.30 ± 0.01 nM potency against DPDPE and deltorphin II in the [35S]GTPx3b3;S functional assay, respectively. The rank order of potency of putative x3b4;1- and x3b4;2-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human x3b4;-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)-x3b2;-MePhe-Phe-OH is a highly potent and x3b4;-opioid specific antagonist both in vivo and in vitro. However, the putative x3b4;1- and x3b4;2-opioid receptors could not be unequivocally distinguished in vitro.