title: A flow-through fluorescence polarization detection system for measuring GPCR-mediated modulation of cAMP production.
abstract: A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z' factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H(3) G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC(50) values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements.
authors: LACDR-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, The Netherlands.
authors: Jeroen
authors: J
authors: Kool
authors: André
authors: A
authors: van Marle
authors: Saskia
authors: S
authors: Hulscher
authors: Maurice
authors: M
authors: Selman
authors: Dick J
authors: DJ
authors: van Iperen
authors: Klaas
authors: K
authors: van Altena
authors: Michel
authors: M
authors: Gillard
authors: Remko A
authors: RA
authors: Bakker
authors: Hubertus
authors: H
authors: Irth
authors: Rob
authors: R
authors: Leurs
authors: Nico P E
authors: NP
authors: Vermeulen
mesh_terms: D000818:Animals; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D009711:Nucleotides / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D009711:Nucleotides; D043562:Receptors, G-Protein-Coupled; D000242:Cyclic AMP