Murase H, Yasuda K, Mercado-Asis L B, Mori A, Shimada T, Mune T, Morita H, Noritake N, Yamakita N, Miura K
Third Department of Internal Medicine, Gifu University School of Medicine, Japan.
J Steroid Biochem Mol Biol. 1991 Mar;38(3):331-7. doi: 10.1016/0960-0760(91)90104-d.
To verify the aldosterone amplifying action of 19-hydroxyandrostenedione (19-OH-AD), we investigated [3H]aldosterone and [3H]19-OH-AD binding to type I (mineralocorticoid) receptor in the renal cytosol of adrenalectomized and ovariectomized rat, and human mononuclear leucocytes (MNL). In the [3H]aldosterone binding study, the cytosol was incubated with [3H]aldosterone and 200-fold RU28362 (11 beta,17 beta-dihydroxy-6-methyl,17 alpha-(1-propynyl)-androsta-1,4,6- trien-3-one), a pure glucocorticoid, with or without 19-OH-AD. Scatchard plots of [3H]aldosterone binding to cytosol with 0.2 or 20 nM 19-OH-AD or without 19-OH-AD were linear. Dissociation constants (Kd) and maximum bindings (Bmax) without 19-OH-AD, and with 0.2 and 20 nM 19-OH-AD were: 0.71 +/- 0.03 nM and 23.0 +/- 3.4 fmol/mg protein (mean +/- SD, n = 3), 0.72 +/- 0.05 nM and 23.1 +/- 2.3 fmol/mg protein (n = 3), and 0.77 +/- 0.04 nM and 22.9 +/- 4.8 fmol/mg protein (n = 3), respectively. 19-OH-AD did not significantly change the Kd and Bmax of [3H]aldosterone binding. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM [3H]aldosterone bound to cytosol. In human MNL, Scatchard plots of [3H]aldosterone binding with both 0.2 and 20 nM 19-OH-AD and without 19-OH-AD were linear. Kd and Bmax were, respectively, 1.00 nM and 780 sites/cell in the absence of 19-OH-AD, and 1.07 nM and 774 sites/cell in the presence of 0.2 nM 19-OH-AD. Without 19-OH-AD they were, respectively, 0.95 nM and 551 sites/cell, and 1.10 nM and 560 sites/cell with 20 nM 19-OH-AD. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM of [3H]aldosterone bound to MNL. In both tissues, there was no obvious specific binding of [3H]19-OH-AD within the range of 1-60 nM. The above results suggest that the amplifying effect of 19-OH-AD on aldosterone mineralocorticoid action may not occur at the binding site of aldosterone to type I receptor, and that 19-OH-AD itself may not have any direct or indirect mineralocorticoid actions on the steroid receptor-mediated process in the rat kidney and human MNL.
为验证19-羟基雄烯二酮（19-OH-AD）对醛固酮的放大作用，我们研究了[3H]醛固酮和[3H]19-OH-AD与肾上腺切除及卵巢切除大鼠肾细胞溶质以及人单核白细胞（MNL）中I型（盐皮质激素）受体的结合情况。在[3H]醛固酮结合研究中，将细胞溶质与[3H]醛固酮以及200倍量的RU28362（11β,17β-二羟基-6-甲基,17α-(1-丙炔基)-雄甾-1,4,6-三烯-3-酮）（一种纯糖皮质激素）一起孵育，同时设置添加或不添加19-OH-AD的组。[3H]醛固酮与添加0.2或20 nM 19-OH-AD或不添加19-OH-AD的细胞溶质的Scatchard图呈线性。不添加19-OH-AD以及添加0.2和20 nM 19-OH-AD时的解离常数（Kd）和最大结合量（Bmax）分别为：0.71±0.03 nM和23.0±3.4 fmol/mg蛋白质（均值±标准差，n = 3）、0.72±0.05 nM和23.1±2.3 fmol/mg蛋白质（n = 3）以及0.77±0.04 nM和22.9±4.8 fmol/mg蛋白质（n = 3）。19-OH-AD未显著改变[3H]醛固酮结合的Kd和Bmax。高浓度的19-OH-AD可轻微置换与细胞溶质结合的0.2或5 nM [3H]醛固酮。在人MNL中，[3H]醛固酮与添加0.2和20 nM 19-OH-AD以及不添加19-OH-AD的Scatchard图呈线性。不添加19-OH-AD时Kd和Bmax分别为1.00 nM和780个位点/细胞，添加0.2 nM 19-OH-AD时分别为1.07 nM和774个位点/细胞。不添加19-OH-AD时分别为0.95 nM和551个位点/细胞，添加20 nM 19-OH-AD时分别为1.10 nM和560个位点/细胞。高浓度的19-OH-AD可轻微置换与MNL结合的0.2或5 nM [3H]醛固酮。在这两种组织中，在1 - 60 nM范围内[3H]19-OH-AD均无明显特异性结合。上述结果表明，19-OH-AD对醛固酮盐皮质激素作用的放大效应可能并非发生在醛固酮与I型受体的结合位点，且19-OH-AD本身可能对大鼠肾脏和人MNL中类固醇受体介导的过程没有任何直接或间接的盐皮质激素作用。
