title: cGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland.
abstract: The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG.
authors.affiliation: Department of Gastroenterology and Hepatology, Erasmus University Medical Center, Rotterdam, The Netherlands;
authors.forename: Hugo R
authors.initials: HR
authors.lastname: De Jonge
authors.forename: Ben C
authors.initials: BC
authors.lastname: Tilly
authors.forename: Boris M
authors.initials: BM
authors.lastname: Hogema
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Pfau
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Kelley
authors.forename: Megan H
authors.initials: MH
authors.lastname: Kelley
authors.forename: August M
authors.initials: AM
authors.lastname: Melita
authors.forename: Montana T
authors.initials: MT
authors.lastname: Morris
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Viola
authors.forename: John N
authors.initials: JN
authors.lastname: Forrest
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000378:metabolism*; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000037:antagonists & inhibitors / Q000378:metabolism; D062967:Cyclic GMP-Dependent Protein Kinase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000378:metabolism*; D004284:Dogfish / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D005260:Female; D029941:Fish Proteins / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors / Q000378:metabolism; D015640:Ion Channel Gating; D008297:Male; D020098:Natriuretic Peptide, C-Type / Q000378:metabolism*; D058987:Phosphodiesterase 3 Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism; D012491:Salt Gland / Q000187:drug effects / Q000201:enzymology*; D015290:Second Messenger Systems; D013997:Time Factors
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D002712:Chlorides; D029941:Fish Proteins; D058987:Phosphodiesterase 3 Inhibitors; D047428:Protein Kinase Inhibitors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D020098:Natriuretic Peptide, C-Type; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D062967:Cyclic GMP-Dependent Protein Kinase Type II; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases; D017461:Receptors, Atrial Natriuretic Factor; C073616:atrial natriuretic factor receptor C; D006152:Cyclic GMP
keywords: C-type natriuretic peptide; CFTR; CNP; cyclic GMP; cystic fibrosis transmembrane conductance regulator; phosphodiesterase; shark rectal gland