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.
Naunyn Schmiedebergs Arch Pharmacol. 2012 Jun;385(6):651-6. doi: 10.1007/s00210-012-0728-0.
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.
尿鸟素 II（U-II）是尿鸟素 II 受体（UT）的肽激动剂。已经发现，假定的 UT 拮抗剂，尿肽和 UFP-803，在一系列测定中具有不同的功效。我们使用 siRNA 介导的 RNA 干扰来探测这些配体与 U-II 相比的功效。在相同的细胞背景下，使用相同的偶联机制敲除人 UT，允许探测相对功效。使用 siPORT 胺转染试剂将 1,110 fmol/mg 蛋白的人 UT（CHOhUT）稳定表达的 CHO 细胞转染 s194454、s194455（UT 靶向）或阴性对照 siRNA。48 小时后，使用定量 PCR 在双探针测定格式中评估沉默。通过 NOVOstar 平板读数器测量 Fura-2 加载细胞中的 [Ca2+]i 来评估沉默的功能后果。用 s194455 沉默的效果大于用 s194454 沉默的效果（分别在 10−7 M 时，UT mRNA 的敲低率为 93.5±2.8%和 73.0±2.5%，p00.006）。s194455 和 s194454 以相等的效力敲低 UT mRNA 表达（EC50 为 1.38 和 0.45 nM）。阴性对照不影响 UT mRNA 表达。U-II（10−6M）增加 [Ca2+]i 630±69、402±49 和 190±14 nM，尿肽（10−6 M）增加 [Ca2+]i 408±55、191±40 和 131±10 nM，UFP-803（10−6 M）增加 [Ca2+]i 134±23、83±11 和 53±3 nM，对于阴性对照 siRNA、s194454 和 s194455 分别为。我们已经证明了 UT mRNA 的沉默和三种 UT 配体的绝对功效降低。然而，我们无法确定尿肽和 UFP-803 的相对功效有任何变化。这可能是由于起始表达高和保留受体/偶联储备。
