title: Development of an improved IP(1) assay for the characterization of 5-HT(2C) receptor ligands.
abstract: The 5-hydroxytryptamine 2C (5-HT(2C)) receptor is a member of the serotonin 5-HT(2) subfamily of G-protein-coupled receptors signaling predominantly via the phospholipase C (PLC) pathway. Stimulation of phosphoinositide (PI) hydrolysis upon 5-HT(2C) receptor activation is traditionally assessed by measuring inositol monophosphate (IP(1)) using time-consuming and labor-intensive anion exchange radioactive assays. In this study, we have developed and optimized a cellular IP(1) assay using homogeneous time-resolved fluorescence (HTRF), a fluorescence resonance energy transfer (FRET)-based technology (Cisbio; Gif sur Yvette, France). The measurement is simple to carry out without the cumbersome steps associated with radioactive assays and may therefore be used as an alternative tool to evaluate PI hydrolysis activated by 5-HT(2C) agonists. In Chinese hamster ovary (CHO) cells stably expressing 5-HT(2C) receptors, characterization of 5-HT(2C) agonists with the HTRF platform revealed a rank order of potency (EC(50), nM) comparable to that from intracellular calcium mobilization studies measured by the fluorometric imaging plate reader (FLIPR). A similar rank order of potency was seen with conventional radioactive PI assay with the exception of 5-HT. Lastly, the new assay data correlated better with agonist-induced calcium responses in FLIPR (R(2) = 0.78) than with values determined by radioactive IP(1) method (R(2) = 0.64). Our study shows that the HTRF FRET-based assay detects IP(1) with good sensitivity and may be streamlined for high-throughput (HTS) applications.
authors.affiliation: Department of Neuroscience, Wyeth Research, 865 Ridge Road, Monmouth Junction, NJ 08852, USA. zhangj@wyeth.com
authors.forename: Jean Y
authors.initials: JY
authors.lastname: Zhang
authors.forename: Dianne M
authors.initials: DM
authors.lastname: Kowal
authors.forename: Stanley P
authors.initials: SP
authors.lastname: Nawoschik
authors.forename: John
authors.initials: J
authors.lastname: Dunlop
authors.forename: Mark H
authors.initials: MH
authors.lastname: Pausch
authors.forename: Ravikumar
authors.initials: R
authors.lastname: Peri
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D008024:Ligands; D044405:Receptor, Serotonin, 5-HT2C / Q000502:physiology*
chemical_list: D007295:Inositol Phosphates; D008024:Ligands; D044405:Receptor, Serotonin, 5-HT2C; D002118:Calcium