In vitro siRNA-mediated knockdown of the UT receptor: implications of density on the efficacy of a range of UT ligands.
Urotensin-II (U-II) is the peptide agonist for the U-II receptor (UT). Putative UT antagonists, urantide and UFP-803, have been found to have variable efficacy in a range of assays. We have used siRNA-mediated RNA interference to probe the efficacy of these ligands compared to U-II. Knockdown of human UT occurs in the same cellular background with the same coupling machinery allowing relative efficacy to be probed. CHO cells stably expressing 1,110 fmol/mg protein of human UT (CHOhUT) were transfected with s194454, s194455 (UT-targeting), or a negative control siRNA using siPORT amine transfection reagent. After 48 h,silencing was assessed using quantitative PCR in a duplex assay format. Functional consequences of silencing were assessed by measuring [Ca2+]i in Fura-2 loaded cells using the NOVOstar plate reader. Silencing with s194455 was greater than that with s194454 (93.5±2.8% and 73.0±2.5%knockdown of UT mRNA respectively at 10−7 M, p00.006).Both s194455 and s194454 knocked down UT mRNA expression with equal potency (EC50 1.38 and 0.45 nM). The negative control did not affect UT mRNA expression. U-II(10−6M) increased [Ca2+]i 630±69, 402±49 and 190±14nM,urantide (10−6 M) increased [Ca2+]i 408±55, 191±40, and 131±10 nM and UFP-803 (10−6 M) increased [Ca2+]i 134±23, 83±11 and 53±3nM for negative control siRNA, s194454 and s194455, respectively.We have demonstrated silencing of UT mRNA and a reduction of absolute efficacy of three UT ligands. However, we were unable to resolve any changes in relative efficacy for urantide and UFP-803. This is likely to result from a high starting expression and retention of a receptor/coupling reserve.
University Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group) and Leicester NIHR Cardiovascular Biomedical Research Unit, Division of Anaesthesia, Critical Care and Pain Management,University of Leicester, Leicester Royal Infirmary, Leicester, UK.
Benjamin D
BD
Hunt
Leong L
LL
Ng
David G
DG
Lambert
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D055785:Gene Knockdown Techniques; D006801:Humans; D010446:Peptide Fragments / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014579:Urotensins / Q000494:pharmacology*
D010446:Peptide Fragments; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C121697:UTS2R protein, human; D014579:Urotensins; C492079:urotensin II (4-11), Pen(5)-Trp(7)-Orn(8)-; C029677:urotensin II; D002118:Calcium