Chou Y C, Luttge W G
Department of Neuroscience, University of Florida College of Medicine, Gainesville 32610.
Brain Res. 1988 Feb 2;440(1):67-78. doi: 10.1016/0006-8993(88)91159-6.
Exchange assays have often been used to quantitate steroid receptors when endogenous ligands are present; however, there are no reports of their successful application to activated glucocorticoid-Type II receptor complexes. In addition to investigating the reasons for this failure, the present study also examined the effects of progesterone on glucocorticoid dissociation from, and reassociation with unactivated and activated Type II receptors. Molybdate-stabilized brain cytosol from adrenal-ovariectomized mice was incubated with [3H]dexamethasone ( +/- [1H]DEX) for 40 h at 0 degree C. Afterwards free steroid was removed on Sephadex G-25 columns in the presence (unactivated receptors) or absence (activated receptors) of molybdate. Activation, as measured by DNA-cellulose binding, was achieved by incubating molybdate-free cytosol at 22 degrees C for 20 min followed by G-25 filtration in the presence of molybdate. The rates of dissociation and reassociation were then measured by incubating cytosol with [1H]triamcinolone acetonide (TA) or [3H]TA ( +/- [1H]TA) at 12 degrees C. An exchange assay was also employed in which cytosol was incubated first with [1H]DEX for 40 h at 0 degree C followed by bound-free steroid separations and 12 degrees C incubations with [3H]TA ( +/- [1H]TA). Both approaches revealed that even though activation reduced the rate of DEX dissociation from Type II receptors by 40%, it eliminated the ability of the newly unoccupied receptors to rebind glucocorticoid. Adding [1H]progesterone to occupied receptor preparations increased dissociation rate constants by nearly 3-fold, for both unactivated and activated Type II receptors. Since [1H]TA failed to prevent this effect, progesterone appears to act at an allosteric site(s) which cannot be occupied by glucocorticoids. Exchange assays revealed that progesterone-facilitated dissociation increased the rate of glucocorticoid rebinding to unactivated, but not activated Type II receptors. These results suggest that spontaneous and progesterone-facilitated termination of glucocorticoid genomic actions could be mediated by steroid dissociation since unoccupied activated Type II receptors do not rebind agonist steroid.
当存在内源性配体时，交换分析常常被用于定量甾体受体；然而，尚无关于其成功应用于活化的糖皮质激素Ⅱ型受体复合物的报道。除了探究这种失败的原因，本研究还考察了孕酮对糖皮质激素从未活化和活化的Ⅱ型受体上解离及重新结合的影响。将来自肾上腺-卵巢切除小鼠的钼酸盐稳定化脑胞质溶胶与[³H]地塞米松（±[¹H]DEX）在0℃孵育40小时。之后，在存在钼酸盐（未活化受体）或不存在钼酸盐（活化受体）的情况下，通过Sephadex G-25柱去除游离甾体。通过在22℃将无钼酸盐的胞质溶胶孵育20分钟，然后在存在钼酸盐的情况下进行G-25过滤，以DNA-纤维素结合来衡量活化情况。然后通过在12℃将胞质溶胶与[¹H]曲安奈德（TA）或[³H]TA（±[¹H]TA）孵育来测量解离和重新结合的速率。还采用了一种交换分析，其中首先将胞质溶胶与[¹H]DEX在0℃孵育40小时，随后进行结合态-游离甾体分离，并在12℃与[³H]TA（±[¹H]TA）孵育。两种方法均表明，尽管活化使DEX从Ⅱ型受体上的解离速率降低了40%，但它消除了新空出的受体重新结合糖皮质激素的能力。向占据受体的制剂中添加[¹H]孕酮，对于未活化和活化的Ⅱ型受体，解离速率常数均增加了近3倍。由于[¹H]TA未能阻止这种效应，孕酮似乎作用于糖皮质激素无法占据的变构位点。交换分析表明，孕酮促进的解离增加了糖皮质激素与未活化但非活化的Ⅱ型受体重新结合的速率。这些结果表明，糖皮质激素基因组作用的自发终止和孕酮促进的终止可能由甾体解离介导，因为未占据的活化Ⅱ型受体不会重新结合激动剂甾体。
