adenosine-5--(n-ethylcarboxamide) and bis(1-3-diethylthiobarbiturate)trimethineoxonol

Adenosine-3',5'-cyclic monophosphate (cAMP) conveys the signals from G-protein coupled receptors (GPCRs) and regulates a variety of downstream cellular events. However, there are few robust assays available for measuring cAMP in live cells. Most of the existing cAMP assays require cell lysis and/or have relatively low throughput. We report a live cell-based cAMP assay that has been developed to record the real-time changes in intracellular cAMP. By employing a mutated cyclic-nucleotide-gated ion channel (CNGC) as the cAMP biosensor, the change of cAMP level is coupled to the change of transmembrane potential that is measured through a new fluorescent membrane potential (MP)-sensitive dye, HLB 021-152. We have successfully used HLB 021-152 for homogeneously monitoring cAMP stimulations in live cells under both serum-containing and serum-free environments. Upon stimulating the endogenous or heterogenous GPCRs on CNGC-cloned human embryonic kidney 293 cells with agonists, the fluorescence signal of HLB 021-152 increases rapidly. It has much greater assay dynamic range than DiSBAC2(3), the existing "gold standard" dye for measuring cellular MP. This new MP dye can be readily formulated for high throughput screening of GPCR modulators either with serum or without serum.

Topics: 3T3 Cells; Adenosine-5'-(N-ethylcarboxamide); Animals; Barbiturates; Biosensing Techniques; Cell Line; Cyclic AMP; Cyclic Nucleotide-Gated Cation Channels; Cytoplasm; Dose-Response Relationship, Drug; Fluorescence; Fluorescent Dyes; Humans; Ion Channels; Isoxazoles; Kinetics; Membrane Potentials; Mice; Microscopy, Fluorescence; Molecular Structure; Parathyroid Hormone; Peptide Fragments; Potassium Chloride; Receptors, G-Protein-Coupled; Receptors, Prostaglandin E; Thiobarbiturates; Time Factors