Differential regulation of natriuretic peptide receptors on ciliary body epithelial cells.
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.
Crook
R B
RB
Beckman Vision Center, Department of Ophthalmology, Box 0730, University of California, San Francisco, San Francisco, CA 94143, USA.
Chang
A T
AT
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
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