title: Differential regulation of natriuretic peptide receptors on ciliary body epithelial cells.
abstract: Atrionatriuretic peptide (ANP) lowers intraocular pressure in the eyes of humans and rabbits. We examined the effects of natriuretic peptides on cGMP formation and 125I-labelled-ANP binding to cultured cells derived from ciliary body epithelium, the site of aqueous humour formation in the eye. ANP, brain natriuretic peptide (BNP) and C-natriuretic peptide (CNP) at 1 microM stimulated cGMP formation 8.2(+/-1.2)-fold, 4.8(+/-0.6)-fold and 87.3(+/-12.1)-fold respectively. 125I-ANP bound to intact cells at a single site, with a dissociation constant KD=0.30+/-0.01 nM. BNP was as effective as ANP in displacing 125I-ANP, whereas CNP displaced label with a slightly higher IC50. 125I-ANP binding was displaced >95% by c-ANP, a specific ligand for natriuretic peptide C receptors (NPR-C). Cross-linking of 125I-ANP to cells labelled predominantly a protein of Mr 62000. These data suggest that 125I-ANP binding was primarily to NPR-C, whereas cGMP stimulation occurred primarily via natriuretic peptide B receptors (NPR-B). Vasopressin and histamine, both activators of the inositol phosphate/diacylglycerol phosphate pathway in non-pigmented ciliary epithelial cells, inhibited CNP stimulation of guanylate cyclase (NPR-B) and 125I-ANP binding (NPR-C) by 30-38%. Inhibition was mimicked by PMA, dioctanoylglycerol and phorbol didecanoate, whereas 4alpha phorbol didecanoate had no effect. Staurosporine and bisindolylmaleimide both blocked inhibition of 125I-ANP binding and cGMP formation by PMA. These results suggest that protein kinase C (PKC) down-regulates both NPR-B and NPR-C. PKC down-regulation of NPR-B varied inversely with CNP concentration. Inhibition by 1 microM PMA was 30.6(+/-4.0)% with 500 nM CNP, but 83.4(+/-8.8)% with 10 nM CNP, indicating that increasing CNP could partially overcome inhibition by PMA. Since extracellular CNP levels were not affected by PKC activation, the effect of PKC on NPR-B is best explained as a reduction in NPR-B affinity for CNP. NPR-C measured as 125I-ANP binding was likewise reduced 36.4(+/-5.1)% by exposure to PMA. In contrast with NPR-B inhibition, however, inhibition of NPR-C was due largely to a reduction in the number of receptor binding sites per cell rather than a reduction in receptor affinity for ligand. The data therefore suggest that both NPR-B and NPR-C are down-regulated by PKC, but that the mechanisms of down-regulation of the two receptors are different.
authors.affiliation: Beckman Vision Center, Department of Ophthalmology, Box 0730, University of California, San Francisco, San Francisco, CA 94143, USA.
authors.forename: R B
authors.initials: RB
authors.lastname: Crook
authors.forename: A T
authors.initials: AT
authors.lastname: Chang
mesh_terms: D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism / Q000494:pharmacology*; D001667:Binding, Competitive; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D015536:Down-Regulation; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005333:Fetus; D006162:Guanylate Cyclase / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D020097:Natriuretic Peptide, Brain; D020098:Natriuretic Peptide, C-Type; D009419:Nerve Tissue Proteins / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000494:pharmacology*; D011817:Rabbits; D017461:Receptors, Atrial Natriuretic Factor / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
chemical_list: D009419:Nerve Tissue Proteins; D011506:Proteins; D014667:Vasopressins; D020097:Natriuretic Peptide, Brain; D020098:Natriuretic Peptide, C-Type; D006632:Histamine; D009320:Atrial Natriuretic Factor; D002217:Carbachol; D011493:Protein Kinase C; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; D006152:Cyclic GMP; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate