Crook R B, Chang A T
Beckman Vision Center, Department of Ophthalmology, Box 0730, University of California, San Francisco, San Francisco, CA 94143, USA.
Biochem J. 1997 May 15;324 ( Pt 1)(Pt 1):49-55. doi: 10.1042/bj3240049.
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.
心钠素（ANP）可降低人和兔眼的眼压。我们研究了利钠肽对环磷酸鸟苷（cGMP）生成以及125I标记的ANP与来源于睫状体上皮（眼房水生成部位）的培养细胞结合的影响。1微摩尔的ANP、脑钠素（BNP）和C型钠尿肽（CNP）分别刺激cGMP生成8.2（±1.2）倍、4.8（±0.6）倍和87.3（±12.1）倍。125I-ANP在完整细胞上的结合位点单一，解离常数KD = 0.30±0.01纳摩尔。BNP在置换125I-ANP方面与ANP效果相同，而CNP置换标记物时的半数抑制浓度（IC50）略高。c-ANP（一种利钠肽C受体（NPR-C）的特异性配体）可使125I-ANP的结合位移>95%。125I-ANP与细胞的交联主要标记了一种分子量为62000的蛋白质。这些数据表明，125I-ANP的结合主要是与NPR-C结合，而cGMP的刺激主要通过利钠肽B受体（NPR-B）发生。血管加压素和组胺都是无色素睫状上皮细胞中肌醇磷酸/二酰基甘油磷酸途径的激活剂，它们可使CNP对鸟苷酸环化酶（NPR-B）的刺激作用和125I-ANP的结合（NPR-C）受到30% - 38%的抑制。佛波酯（PMA）、二辛酰甘油和十四酰佛波醇乙酯可模拟这种抑制作用，而4α-十四酰佛波醇乙酯则无作用。星形孢菌素和双吲哚马来酰胺均可阻断PMA对125I-ANP结合和cGMP生成的抑制作用。这些结果表明，蛋白激酶C（PKC）可下调NPR-B和NPR-C。PKC对NPR-B的下调与CNP浓度呈反比。1微摩尔PMA的抑制作用在500纳摩尔CNP时为30.6（±4.0）%，但在10纳摩尔CNP时为83.4（±8.8）%，这表明增加CNP可部分克服PMA的抑制作用。由于细胞外CNP水平不受PKC激活的影响，PKC对NPR-B的作用最好解释为NPR-B对CNP的亲和力降低。以125I-ANP结合来衡量，NPR-C同样因暴露于PMA而降低了36.4（±5.1）%。然而，与NPR-B的抑制作用不同，NPR-C的抑制主要是由于每个细胞上受体结合位点数量的减少，而不是受体对配体亲和力的降低。因此，数据表明NPR-B和NPR-C均被PKC下调，但两种受体下调的机制不同。
