title: Identification, regulation and anti-proliferative role of the NPR-C receptor in gastric epithelial cells.
abstract: Evidence suggests that functional atrial natriuretic peptide (ANP) receptors occur in surface gastric mucosal epithelial cells. To evaluate functional aspects of ANP in a model of these cells we examined the expression of natriuretic peptide receptors (NPR) subtypes A and C in the non-transformed rat gastric mucosal epithelial cell line RGM1. Transcripts for NPR-A and NPR-C were detected in RGM1 cells by RT-PCR. However, only NPR-C protein was detected by Western blot and immunohistochemical analyses. Specific saturable binding of (125)I-ANP to RGM1 cells revealed a single class of high affinity binding sites (K (d) = 208 +/- 71pM, B (max) = 110,000 +/- 14,000 sites/cell, Hill coefficient = 0.97 +/- 0.05). ANP (IC(50) 130 +/- 47pM), BNP (IC(50) 716 +/- 26 pM), CNP (IC(50) 356 +/- 85pM) and C-ANP (IC(50) 134 +/- 13pM), a specific ligand for NPR-C, effectively displaced (125)I-ANP binding. Cross-linking of (125)I-ANP to cells labeled predominantly a protein of 66,000 Da. These data suggest that (125)I-ANP binding was primarily to NPR-C. ANP and C-ANP inhibited forskolin- and prostaglandin E(2) (PGE(2))-stimulated cAMP in a PTx-sensitive fashion. PGE(2), transforming growth factor-+/-1 (TGF-+/-1), forskolin, 8-bromo-cyclic AMP, and phorbol-12-myristate-13-acetate (PMA) caused a dose-dependent decrease in specific (125)I-ANP binding, whereas epidermal growth factor (EGF), 8-bromo-cyclic GMP and 4+/--phorbol didecanoate had no effect. PGE(2), forskolin, TGF-+/-1 and PMA significantly decreased (125)I-ANP B (max) values, NPR-C protein and steady-state NPR-C transcript levels. H89, a protein kinase A inhibitor, blocked the reduction of NPR-C mRNA produced by both forskolin and PGE(2.) GF109203X, a protein kinase C inhibitor, abolished the PMA-induced decrease in NPR-C transcripts but only partially blocked that produced by TGF-+/-1. RGM1 cells exhibited a dose-dependent decrease in both DNA synthesis and cell proliferation when cultured in the presence of ANP or C-ANP. These findings indicate that RGM1 cells express functional NPR-C receptors that can influence RGM1 cell proliferation and are down-regulated by PGE(2) and TGF-+/-1.
authors.affiliation: Surgery and Research Services, James A. Haley Veterans Administration Hospital, 13000 Bruce B. Downs Boulevard, Tampa, FL 33612-4745, USA. wgower@health.usf.edu
authors.forename: William R
authors.initials: WR
authors.lastname: Gower
authors.forename: Gay M
authors.initials: GM
authors.lastname: Carter
authors.forename: Quentin
authors.initials: Q
authors.lastname: McAfee
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Solivan
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D049109:Cell Proliferation; D005576:Colforsin / Q000494:pharmacology; D015232:Dinoprostone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004847:Epithelial Cells / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005753:Gastric Mucosa / Q000166:cytology / Q000378:metabolism*; D008297:Male; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017461:Receptors, Atrial Natriuretic Factor / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
chemical_list: D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D005576:Colforsin; D009320:Atrial Natriuretic Factor; D011494:Protein Kinases; D000262:Adenylyl Cyclases; D017461:Receptors, Atrial Natriuretic Factor; C073616:atrial natriuretic factor receptor C; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate