title: FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells.
abstract: Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3-6 d.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Börner
authors.forename: Frank
authors.initials: F
authors.lastname: Schwede
authors.forename: Angela
authors.initials: A
authors.lastname: Schlipp
authors.forename: Filip
authors.initials: F
authors.lastname: Berisha
authors.forename: Davide
authors.initials: D
authors.lastname: Calebiro
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
authors.forename: Viacheslav O
authors.initials: VO
authors.lastname: Nikolaev
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D015374:Biosensing Techniques; D002138:Calibration; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D020662:Guanine Nucleotide Exchange Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D032383:Myocytes, Cardiac / Q000378:metabolism; D013961:Thyroid Gland / Q000378:metabolism
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; C482601:Epac protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D000242:Cyclic AMP