Cell and tissue responses of a range of Urotensin II analogs at cloned and native urotensin II receptors. Evidence for coupling promiscuity

Urotensin II (U-II) is the peptide ligand for the G-protein-coupled U-II receptor (UT). U-II has been dubbed “the most potent vasoconstrictor identified to date”. However, in vivo studies with this system are hampered by the paucity of available ligands. Here, we characterise Chinese hamster ovary (CHO) cells expressing the human UT receptor in the following assays; (1) [125I]UII binding, (2) GTPγ[35S] binding, (3) cAMP formation, and (4) intracellular Ca2+. We assess activity of 9 U-II analogues using these paradigms and examine their ability to contract isolated rat aorta. CHOhUT cells bound [125I]UII with a Bmax and Kd of 1,110±70 fmol/mg protein and 742 pM, respectively. hU-II stimulated GTPγ[35S] binding (pEC50 8.38), optimal at low (0.1 μM) GDP concentrations. The hU-II GTPγ[35S] response was partially PTx sensitive and there was a potent (pEC50 9.23) low efficacy (∼20% inhibition) coupling to adenylyl cyclase. In CHOhUT cells hU-II stimulates calcium release from intracellular stores (pEC50 8.80) and calcium influx in a PTx-insensitive manner. In our structure-activity relationship study most ligands acted as full agonists. However, urantide behaved as a partial agonist (pEC50 7.67/pKB 7.55) in GTPγ[35S] binding, a full agonist (pEC50 8.11) for increases in intracellular Ca2+ and a competitive antagonist in the rat aorta bioassay (pKB 8.59). Collectively, these data show promiscuity at high expression and indicate the need for careful multi-assay evaluation of novel U-II analogues. Further modification of urantide, in order to eliminate residual agonist activity and to identify novel ligands for in vivo cardiovascular studies are clearly warranted.