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 I-125-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 mu M stimulated cGMP formation 8.2(+/- 1.2)-fold, 4.8(+/- 0.6)-fold and 87.3(+/- 12.1)-fold respectively. I-125-ANP bound to intact cells at a single site, with a dissociation constant K-D= 0.30 +/- 0.01 nM. BNP was as effective as ANP in displacing I-125- ANP, whereas CNP displaced label with a slightly higher IC50 I-125-ANP binding was displaced > 95% by c-ANP, a specific ligand for natriuretic peptide C receptors (NPR-C). Cross-linking of I-125-ANP to cells labelled predominantly a protein of M(r)62000. These data suggest that I-125-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 I-125-ANP binding (NPR-C) by 30-38 %. Inhibition was mimicked by PMA, dioctanoylglycerol and phorbol didecanoate, whereas 4 alpha phorbol didecanoate had no effect. Staurosporine and bisindolylmaleimide both blocked inhibition of I-125-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 mu M 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 I-125-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.