title: Pharmacological characterization of receptor guanylyl cyclase reporter cell lines.
abstract: Receptor guanylyl cyclases are implicated in a growing number of pathophysiologies and, therefore, represent an important target class for drug development. We report here the generation and pharmacological characterization of three particulate guanylyl cyclase (pGC) reporter cell lines. Plasmid constructs encoding the natriuretic peptide receptors GC-A and GC-B, and the heat-stable enterotoxin receptor GC-C, were stably transfected in a parental reporter cell line expressing a cyclic nucleotide-gated (CNG) cation channel, acting as the biosensor for intracellular cGMP. In our reporter cell lines pGC activity can be monitored in living cells in real-time . By using different natural as well as synthetic receptor ligands of the natriuretic and guanylin peptide families, we show that our reporter assay monitors pGC activity with very high sensitivity. In contrast to previous findings, we could detect significant stimulation of GC-A and GC-B by each of the natriuretic peptides ANP, BNP and CNP. In addition, the clearance receptor ligand Cys-ANF(4-18) and the ANP receptor antagonist Arg-ANF(6-18) were characterized as partial GC-A agonists. The results imply that our novel pGC reporter cell lines are well suited for the characterization of receptor pharmacology and may be used for natural ligand characterization of guanylyl cyclase orphan receptors.
authors.affiliation: Lead Discovery Wuppertal, Bayer HealthCare AG, Pharma Research Center, Aprather Weg 18a, Wuppertal D-42096, Germany. frank.wunder@bayer.com
authors.forename: Frank
authors.initials: F
authors.lastname: Wunder
authors.forename: Annette
authors.initials: A
authors.lastname: Woermann
authors.forename: Andreas
authors.initials: A
authors.lastname: Geerts
authors.forename: Markus
authors.initials: M
authors.lastname: Milde
mesh_terms: D000818:Animals; D009320:Atrial Natriuretic Factor / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D006152:Cyclic GMP / Q000096:biosynthesis; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D050705:Receptors, Guanylate Cyclase-Coupled / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D009320:Atrial Natriuretic Factor; D050705:Receptors, Guanylate Cyclase-Coupled; D006152:Cyclic GMP