De Léan A, Gutkowska J, McNicoll N, Schiller P W, Cantin M, Genest J
Life Sci. 1984 Dec 3;35(23):2311-8. doi: 10.1016/0024-3205(84)90522-8.
We have recently shown that synthetic rat atrial natriuretic factor (ANF) directly inhibits mineralocorticoid and glucocorticoid secretion in cultured bovine adrenal cells with a potency of 100 pM. [125I]iodo-ANF was used in the present study to characterize potential receptor sites in bovine zona glomerulosa membranes. ANF binds to a class of high affinity binding sites with a pK of 10.2 and a density of 1.3 pmol/mg protein. Detailed competition curves with ANF document a class of high affinity sites with a pK of 10.2 and also a second class of lower affinity sites with a pK of 8.5. Nonspecific binding amounts to less than 10% of [125I]iodo-ANF binding at concentrations less than 100 pM. High affinity binding of [125I]iodo-ANF is reversible with a half-time of association of 15 minutes at 25 pM and a half-time of dissociation of 140 minutes. Monovalent cations Na, Li and K equipotently enhance [125I]iodo-ANF specific binding. Divalent cations Mg, Ca and Mn also increase [125I]iodo-ANF specific binding, with Mn being the most active cation. No effect of guanine nucleotide could be detected on ANF binding. The binding of [125I]iodo-ANF is very specific and is not inhibited by 1 microM angiotensin II, ACTH, VIP, somatostatin, Leu-enkephalin, dynorphin or by the N-terminal of POMC. The N-terminal fragment ANF-(1-16) is also completely inactive. Reduction of the disulfide bridge of ANF inactivates the peptide. This enabled the development of a highly specific radio-receptor assay for ANF with a minimum detectable dose of 2 femtomoles. The results document the specific receptor involved in the potent inhibitory effect of ANF on adrenal steroidogenesis and indicate that bovine adrenal zonal glomerulosa provide a highly sensitive system for studying the recently discovered atrial natriuretic factor.
我们最近发现，合成的大鼠心房利钠因子（ANF）可直接抑制培养的牛肾上腺细胞中盐皮质激素和糖皮质激素的分泌，其效力为100 pM。本研究使用[125I]碘-ANF来表征牛球状带细胞膜中的潜在受体位点。ANF与一类高亲和力结合位点结合，其pK为10.2，密度为1.3 pmol/mg蛋白质。用ANF绘制的详细竞争曲线显示出一类pK为10.2的高亲和力位点以及另一类pK为8.5的低亲和力位点。在浓度低于100 pM时，非特异性结合量不到[125I]碘-ANF结合量的10%。[125I]碘-ANF的高亲和力结合是可逆的，在浓度为25 pM时，结合半衰期为15分钟，解离半衰期为140分钟。单价阳离子Na、Li和K等效地增强[125I]碘-ANF的特异性结合。二价阳离子Mg、Ca和Mn也增加[125I]碘-ANF的特异性结合，其中Mn是最具活性的阳离子。未检测到鸟嘌呤核苷酸对ANF结合有影响。[125I]碘-ANF的结合非常特异，不受1 microM血管紧张素II、促肾上腺皮质激素、血管活性肠肽、生长抑素、亮氨酸脑啡肽、强啡肽或阿片促黑皮质素原N端的抑制。N端片段ANF-(1-16)也完全无活性。ANF二硫键的还原使该肽失活。这使得能够开发出一种高度特异性的ANF放射受体测定法，最低可检测剂量为2飞摩尔。这些结果证明了参与ANF对肾上腺类固醇生成强效抑制作用的特异性受体，并表明牛肾上腺球状带为研究最近发现的心房利钠因子提供了一个高度敏感的系统。
