Please use this identifier to cite or link to this item: http://hdl.handle.net/2381/9284
Title: In Vitro Manipulation of Urotensin-II Receptor Expression: Implications of Receptor Density on Ligand Efficacy
Authors: Hunt, Benjamin Dean
Supervisors: Lambert, David
Ng, Leong
Award date: 1-Apr-2011
Presented at: University of Leicester
Abstract: Many urotensin-II (UT) receptor ligands characterised to date exhibit assay-dependent paradoxical agonist / antagonist properties. This activity is likely to be due to differences in UT receptor density between assays, especially between in vivo and in vitro systems. The present study characterised the effect of UT receptor density on the absolute and relative efficacies of experimental UT ligands (urantide and UFP-803). Two in vitro models were produced allowing the density of UT to be controlled in the presence of a fixed cellular background. 1. Ecdysone-inducible Chinese hamster ovary (CHO) cell-line, allowing UT transcription to be induced by a nuclear steroid hormone, (pon-A). 2. UT-targeting siRNAs, allowing UT translation to be inhibited in CHO cells expressing the human recombinant UT receptor (CHOhUT). The production and characterisation of the first of these models was problematic due to significant expression of UT in the absence of pon-A, therefore affording no advantage over classical in vitro systems. Two UT-targeting siRNAs were transfected into CHOhUT and were shown to knockdown UT expression by 94 & 73% at the mRNA level and 64 & 40% at the functional level. This model has allowed us to control UT receptor density in the presence of a fixed cellular background. We have shown that decreasing the density of UT in CHOhUT reduces the absolute efficacies of experimental UT ligands (urantide and UFP-803). We attribute this effect to reduced mass of ligand-receptor complexes. Reducing the density of UT in CHOhUT does not affect the relative efficacies of the same ligands. We attribute this effect to the absence of a UT receptor reserve over the range of UT densities used. A better understanding of system-dependent efficacy is crucial for effective use of UT ligands in the clinical setting. The technologies employed in this thesis represent an important avenue for future work.
Links: http://hdl.handle.net/2381/9284
Type: Thesis
Level: Doctoral
Qualification: PhD
Appears in Collections:Theses, Dept. of Cardiovascular Sciences
Leicester Theses

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