title: Cell and tissue responses of a range of Urotensin II analogs at cloned and native urotensin II receptors. Evidence for coupling promiscuity.
abstract: 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) [(125)I]U-II binding, (2) GTPgamma[(35)S] binding, (3) cAMP formation, and (4) intracellular Ca(2+). We assess activity of 9 U-II analogues using these paradigms and examine their ability to contract isolated rat aorta. CHO(hUT) cells bound [(125)I]U-II with a B (max) and K (d) of 1,110+/-70 fmol/mg protein and 742 pM, respectively. hU-II stimulated GTPgamma[(35)S] binding (pEC(50) 8.38), optimal at low (0.1 muM) GDP concentrations. The hU-II GTPgamma[(35)S] response was partially PTx sensitive and there was a potent (pEC(50) 9.23) low efficacy ( approximately 20% inhibition) coupling to adenylyl cyclase. In CHO(hUT) cells hU-II stimulates calcium release from intracellular stores (pEC(50) 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 (pEC(50) 7.67/pK(B) 7.55) in GTPgamma[(35)S] binding, a full agonist (pEC(50) 8.11) for increases in intracellular Ca(2+) and a competitive antagonist in the rat aorta bioassay (pK(B) 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.
authors.affiliation: University Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE1 5WW, UK.
authors.forename: Wei
authors.initials: W
authors.lastname: Song
authors.forename: John
authors.initials: J
authors.lastname: McDonald
authors.forename: Valeria
authors.initials: V
authors.lastname: Camarda
authors.forename: Girolamo
authors.initials: G
authors.lastname: Calo
authors.forename: Remo
authors.initials: R
authors.lastname: Guerrini
authors.forename: Erika
authors.initials: E
authors.lastname: Marzola
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Thompson
authors.forename: David J
authors.initials: DJ
authors.lastname: Rowbotham
authors.forename: David G
authors.initials: DG
authors.lastname: Lambert
mesh_terms: D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D005576:Colforsin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D037342:Pertussis Toxin / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014579:Urotensins / Q000494:pharmacology*
chemical_list: D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D014579:Urotensins; C473142:Uts2r protein, rat; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C029677:urotensin II; D000242:Cyclic AMP; D037342:Pertussis Toxin; D002118:Calcium