Identification and pharmacological characterization of native, functional human urotensin-II receptors in rhabdomyosarcoma cell lines.

I 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 [I-125]hU-II binding sites. 2 UT mRNA (Northern blot, PCR) and protein (immunocytochemistry) were evident in human skeletal muscle tissue and cells. 3 [I-125]hU-II bound to a homogenous population of high-affinity, saturable (K-d 67.0 +/- 11.8 pM, B-max 9687 +/- 843 sites cell(-1)) receptors in the skeletal muscle (rhabdomyosarcoma) cell line SJRH30. Radiolabel was characteristically slow to dissociate (less than or equal to 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), K-d 74 +/- 8 pM). 4 Consistent with the profile recorded in human recombinant UT-HEK293 cells, [I-125]hU-II binding to SJRH30 cells was selectively displaced by both mammalian and fish U-II isopeptides (K(i)s 0.5 +/- 0.1-1.2 +/- 0.3 nm) and related analogues (hU-II[4-11]>[Cys(5,10)]Acm hU-II; K(i)s 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.