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.