Abstract
Background and purpose:The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties of fluorescent agonists and antagonists.Experimental approach:Chinese hamster ovary cells stably expressing the human adenosine A1 receptor and a cyclic 3′,5′ adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with whole cell [3H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5′ (N-ethylcarboxamido) adenosine (NECA).Key results:Derivatives of NECA and XAC with different fluorophores, but equivalent linker length, showed significant differences in their binding properties to the adenosine A1 receptor. The BODIPY 630/650 derivatives had the highest affinity. Linker length also affected the pharmacological properties, depending on the fluorophore used. Particularly in fluorescent agonists, higher agonist potency could be achieved with large or small linkers for dansyl and BODIPY 630/650 derivatives, respectively.Conclusions and implications:The pharmacology of a fluorescent ligand was critically influenced by both the fluorophore and the associated linker. Furthermore, our data strongly suggest that the physicochemical properties of the fluorophore/linker pairing determine where in the environment of the target receptor the fluorophore is placed, and this, together with the environmental sensitivity of the resulting fluorescence, may finally decide its utility as a fluorescent probe.This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x
Highlights
Adenosine A1 receptors are G protein-coupled receptors (GPCRs) that signal via Gi/o proteins to inhibit adenylyl cyclase activity and reduce the cellular levels of the second messenger, cyclic 3′,5′ adenosine monophosphate
Experimental approach: Chinese hamster ovary cells stably expressing the human adenosine A1 receptor and a cyclic 3′,5′ adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with whole cell [3H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5′ (N-ethylcarboxamido) adenosine (NECA)
In order to systematically test the effect of fluorophore, linker length and composition on ligand pharmacology, we synthesized a number of fluorescent derivatives of the archetypal adenosine A1-receptor agonist, NECA and the corresponding antagonist, XAC (Figures 1 and 2, Table 1; compounds 1–21)
Summary
Adenosine A1 receptors are G protein-coupled receptors (GPCRs) that signal via Gi/o proteins to inhibit adenylyl cyclase activity and reduce the cellular levels of the second messenger, cyclic 3′,5′ adenosine monophosphate (cyclic AMP; Libert et al, 1992; Olah and Styles, 1995; Fredholm et al, 2001). Our current understanding of the molecular pharmacology of the adenosine A1-receptor has been deduced primarily from the use of radioligand binding techniques for studying ligandreceptor interactions and the measurement of intracellular second messenger generation. These experiments require large numbers of cells, and the pharmacological parameters that are deduced from them represent the average for the cell population. The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties of fluorescent agonists and antagonists. British Journal of Pharmacology (2010) 159, 772–786; doi:10.1111/j.1476-5381.2009.00488.x; published online 26 January 2010
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