Development of a high-throughput assay for monitoring cAMP levels in cardiac ventricular myocytes.
G-protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptors involved in cell signal transduction. Many of these GPCRs convey their pharmacological actions by regulating intracellular levels of 3',5'-cyclic adenosine monophosphate (cAMP). Although the heart expresses more than 100 GPCRs, drug agonists for approximately one third of these GPCRs have not been identified. The goal of this project was to initiate the development of a high-throughput screening assay for monitoring cAMP in the heart. Neonatal rat cardiac ventricular myocytes were isolated and cultured on coverslips (whole-cell patch clamp recording) or in 96-well plates (fluorescent imaging plate reader measurements). Cells were infected with adenovirus expressing either beta-galactosidase (AdLacZ) or a mutant cyclic nucleotide-gated (CNG) channel containing the double mutation C460W/E583M (AdCNG). Addition of 2 microM forskolin along with 100 microM 3-isobutyl-1-methylxanthine, to increase intracellular cAMP, activated a cation current in myocytes infected with the AdCNG. In myocytes loaded with the fluorescent Ca indicator Fluo-4, stimulation with forskolin, epinephrine, norepinephrine, or the beta-adrenergic receptor agonist isoproterenol increased the fluorescent signal indicative of Ca influx through the CNG channel. In conclusion, CNG channels are readily expressed in cultured cardiac myocytes and may be utilized in high-throughput screening assays of intracellular cAMP.
Walsh
Kenneth B
KB
Department of Pharmacology, Physiology and Neuroscience, School of Medicine, University of South Carolina, Columbia, SC 29208, USA. walsh@med.sc.edu
Rich
Thomas C
TC
Coffman
Zachary J
ZJ
D000256:Adenoviridae / Q000201:enzymology / Q000235:genetics; D000814:Aniline Compounds / Q000737:chemistry; D000818:Animals; D000831:Animals, Newborn; D015374:Biosensing Techniques / Q000379:methods*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000032:analysis* / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D014966:Xanthenes / Q000737:chemistry; D001616:beta-Galactosidase / Q000235:genetics
D000814:Aniline Compounds; D054815:Cyclic Nucleotide-Gated Cation Channels; C409648:Fluo 4; D005456:Fluorescent Dyes; D014966:Xanthenes; D000242:Cyclic AMP; D001616:beta-Galactosidase