A novel PDE2A reporter cell line: characterization of the cellular activity of PDE inhibitors.
We report here the generation and pharmacological characterization of a phosphodiesterase 2A (PDE2A) reporter cell line. Human PDE2A was stably transfected in a parental cell line expressing the atrial natriuretic peptide (ANP) receptor and the cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the biosensor for intracellular cGMP. In this reporter cell line, cGMP levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the CNG channel. By using different PDE inhibitors, we could show that our PDE2A reporter assay specifically monitors PDE2A inhibition with high sensitivity. In the absence of ANP stimulation, the PDE2A selective inhibitors EHNA, BAY 60-7550 and PDP did not increase basal luminescence levels in this experimental setting. However, in combination with ANP, these inhibitors stimulated luminescence signals and induced leftward shifts of ANP concentration-response curves. Similar results were obtained when using IBMX, trequinsin and dipyridamole, which inhibit PDE2A nonselectively with lower potency. PDP, the most potent PDE2A inhibitor known to date, was found to exhibit much lower cellular activity as anticipated from its biochemical PDE2A inhibitory activity. By cellular uptake and transport studies we could show that PDP's cell permeability is low and that the compound is a substrate for an efflux transporter. Other PDE inhibitors including vinpocetine, milrinone, rolipram, sildenafil, zaprinast, BRL 50481 and BAY 73-6691 did not stimulate luminescence signals on our PDE2A reporter cell line. The results imply that this novel PDE2A reporter assay provides an efficient, high throughput means for the identification and characterization of PDE2A inhibitors.
Wunder
Frank
F
Bayer HealthCare AG, Molecular Screening Technology, Pharma Research Center, D-42096 Wuppertal, Germany. frank.wunder@bayerhealthcare.com
Gnoth
Mark Jean
MJ
Geerts
Andreas
A
Barufe
Daniel
D
D000818:Animals; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D006224:Cricetinae; D006152:Cyclic GMP / Q000096:biosynthesis; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism
D010726:Phosphodiesterase Inhibitors; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; D017461:Receptors, Atrial Natriuretic Factor; D006152:Cyclic GMP