A flow-through fluorescence polarization detection system for measuring GPCR-mediated modulation of cAMP production.
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.
Kool
Jeroen
J
LACDR-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, The Netherlands.
van Marle
André
A
Hulscher
Saskia
S
Selman
Maurice
M
van Iperen
Dick J
DJ
van Altena
Klaas
K
Gillard
Michel
M
Bakker
Remko A
RA
Irth
Hubertus
H
Leurs
Rob
R
Vermeulen
Nico P E
NP
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
D009711:Nucleotides; D043562:Receptors, G-Protein-Coupled; D000242:Cyclic AMP