title: Identification and pharmacological characterization of native, functional human urotensin-II receptors in rhabdomyosarcoma cell lines.
abstract: 1 In an effort to identify endogenous, native mammalian urotensin-II (U-II) receptors (UT), a diverse range of human, primate and rodent cell lines (49 in total) were screened for the presence of detectable [125I]hU-II binding sites. 2 UT mRNA (Northern blot, PCR) and protein (immunocytochemistry) were evident in human skeletal muscle tissue and cells. 3 [(125)I]hU-II bound to a homogenous population of high-affinity, saturable (Kd 67.0+/-11.8 pm, Bmax 9687+/-843 sites cell(-1)) receptors in the skeletal muscle (rhabdomyosarcoma) cell line SJRH30. Radiolabel was characteristically slow to dissociate (< or =15% dissociation 90 min). A lower density of high-affinity U-II binding sites was also evident in the rhabdomyosarcoma cell line TE671 (1667+/-165 sites cell(-1), Kd 74+/-8 pm). 4 Consistent with the profile recorded in human recombinant UT-HEK293 cells, [125I]hU-II binding to SJRH30 cells was selectively displaced by both mammalian and fish U-II isopeptides (Kis 0.5+/-0.1-1.2+/-0.3 nm) and related analogues (hU-II[4-11]>[Cys(5,10)]Acm hU-II; Kis 0.4+/-0.1 and 864+/-193 nm, respectively). 5 U-II receptor activation was functionally coupled to phospholipase C-mediated [Ca2+]i mobilization (EC50 6.9+/-2.2 nm) in SJRH30 cells. 6 The present study is the first to identify the presence of 'endogenous' U-II receptors in SJRH30 and TE671 cells. SJRH30 cells, in particular, might prove to be of utility for (a) investigating the pharmacological properties of hU-II and related small molecule antagonists at native human UT and (b) delineating the role of this neuropeptide in the (patho)physiological regulation of mammalian neuromuscular function.
authors.affiliation: Department of Vascular Biology and Thrombosis, Cardiovascular and Urogenital Center of Excellence for Drug Discovery, GlaxoSmithKline, King of Prussia, PA 19406, USA. steve.a.douglas@gsk.com
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Douglas
authors.forename: Diane
authors.initials: D
authors.lastname: Naselsky
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Ao
authors.forename: Jyoti
authors.initials: J
authors.lastname: Disa
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Herold
authors.forename: Frank
authors.initials: F
authors.lastname: Lynch
authors.forename: Nambi V
authors.initials: NV
authors.lastname: Aiyar
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D015152:Blotting, Northern; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D005260:Female; D020869:Gene Expression Profiling; D006367:HeLa Cells; D006801:Humans; D007028:Hypothalamic Hormones / Q000494:pharmacology; D007150:Immunohistochemistry; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008297:Male; D008543:Melanins / Q000494:pharmacology; D009478:Neuropeptide Y / Q000494:pharmacology; D009481:Neurophysins / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D010907:Pituitary Hormones / Q000494:pharmacology; D011498:Protein Precursors / Q000494:pharmacology; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012208:Rhabdomyosarcoma / Q000378:metabolism* / Q000473:pathology; D019284:Thapsigargin / Q000494:pharmacology; D014579:Urotensins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
chemical_list: C438657:AVP protein, human; D007028:Hypothalamic Hormones; D007457:Iodine Radioisotopes; D008543:Melanins; D009478:Neuropeptide Y; D009481:Neurophysins; D010907:Pituitary Hormones; D011498:Protein Precursors; D043562:Receptors, G-Protein-Coupled; C121697:UTS2R protein, human; D014579:Urotensins; D014667:Vasopressins; C019463:melanin-concentrating hormone; D019284:Thapsigargin; C029676:urotensin I; C029677:urotensin II; D037342:Pertussis Toxin; D015740:Calcitonin Gene-Related Peptide; D002118:Calcium