Sheppard K E
Baker Medical Research Institute, Prahran, Victoria, Australia.
J Steroid Biochem Mol Biol. 1998 Jan;64(1-2):35-42. doi: 10.1016/s0960-0760(97)00135-0.
[3H]Steroid binding in intact colonic crypt cells and cytosol was compared to determine if 11beta hydroxysteroid dehydrogenase (11betaHSD) activity modulates access of corticosterone (B) to both glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). Cytosol from non-adrenalectomized rat colonic crypt cells showed no 11betaHSD activity, and B bound with high affinity to both MR (Kd=0.47+/-0.03 nM; Bmax=177+/-34 fmol/mg protein) and GR (Kd=4.5+/-0.3 nM; Bmax=279+/-40 fmol/mg protein). In contrast, intact colonic crypt cells incubated with 25-30 nM [3H]B for 90 min converted 62% of B to 11-dehydrocorticosterone, with little binding to MR (14+/-3 fmol/mg protein) and GR (22+/-5 fmol/mg protein). When 11betaHSD activity was inhibited with carbenoxolone, and the same concentration of [3H]B used, binding of [3H]B to MR increased 10-fold to 122+/-12 fmol/mg protein, not significantly different from MR levels in colonic crypt cytosol. In contrast, [3H]B binding to GR in intact cells increased only 1.6-fold to 36+/-9 fmol/mg protein, significantly less than to GR in cytosol (212+/-24 fmol/mg protein). Scatchard analysis showed both lower levels of GR and an apparently lower affinity for [3H]B in colonic crypt cells (Kd=31+/-3 nM; Bmax=130+/-21 fmol/mg protein) compared with cytosol (Kd=4.5+/-0.3 nM; Bmax=279+/-40 fmol/mg protein. [3H]Dexamethasone similarly showed an apparently lower affinity and capacity for GR (Kd=8.8+/-1.3 nM; Bmax=232+/-32 fmol/mg protein) in intact cells compared with cytosol (two separate determinations, Kd=2.6 and 2.9 nM; Bmax=369 and 300 fmol/mg protein). In contrast, [3H]aldosterone displayed similar affinity and capacity for MR in both intact cells (Kd=2.0 nM; Bmax=121 fmol/mg protein) and cytosol (Kd=1.5 and 1.4nM; Bmax=115 and 93 fmol/mg protein). These findings demonstrate not only that 11betaHSD modulates binding to both MR and GR in colonic crypt cells, but also that an additional mechanism(s) operating in whole cells but not in cytosol selectively reduces the affinity and capacity of colonic GR for both natural and synthetic ligands.
比较完整结肠隐窝细胞和胞质溶胶中[3H]类固醇结合情况，以确定11β-羟类固醇脱氢酶（11βHSD）活性是否调节皮质酮（B）与糖皮质激素受体（GR）和盐皮质激素受体（MR）的结合。未切除肾上腺大鼠结肠隐窝细胞的胞质溶胶未显示11βHSD活性，B与MR（解离常数Kd = 0.47±0.03 nM；最大结合量Bmax = 177±34 fmol/mg蛋白质）和GR（Kd = 4.5±0.3 nM；Bmax = 279±40 fmol/mg蛋白质）均高亲和力结合。相比之下，用25 - 30 nM [3H]B孵育90分钟的完整结肠隐窝细胞将62%的B转化为11 - 脱氢皮质酮，与MR（14±3 fmol/mg蛋白质）和GR（22±5 fmol/mg蛋白质）的结合很少。当用甘草次酸抑制11βHSD活性并使用相同浓度的[3H]B时，[3H]B与MR的结合增加10倍至122±12 fmol/mg蛋白质，与结肠隐窝细胞胞质溶胶中的MR水平无显著差异。相比之下，完整细胞中[3H]B与GR的结合仅增加1.6倍至36±9 fmol/mg蛋白质明显低于胞质溶胶中的GR（212±24 fmol/mg蛋白质）。Scatchard分析显示，与胞质溶胶（Kd = 4.5±0.3 nM；Bmax = 279±40 fmol/mg蛋白质）相比，结肠隐窝细胞中GR水平较低且对[3H]B的亲和力明显较低（Kd = 31±3 nM；Bmax = 130±21 fmol/mg蛋白质）。[3H]地塞米松同样显示，与胞质溶胶（两次独立测定，Kd = 2.6和2.9 nM；Bmax = 369和3,00 fmol/mg蛋白质）相比，完整细胞中GR的亲和力和结合能力明显较低（Kd = 8.8±1.3 nM；Bmax = 232±32 fmol/mg蛋白质）。相比之下，[3H]醛固酮在完整细胞（Kd = 2.0 nM；Bmax = 121 fmol/mg蛋白质）和胞质溶胶（Kd = 1.5和1.4 nM；Bmax = 115和93 fmol/mg蛋白质）中对MR的亲和力和结合能力相似。这些发现不仅证明11βHSD调节结肠隐窝细胞中与MR和GR的结合，还证明在完整细胞而非胞质溶胶中起作用的另一种机制选择性降低结肠GR对天然和合成配体的亲和力和结合能力。
