Czerwinski S M, McKee E E, Hickson R C
Department of Physical Education, University of Illinois, Chicago 60680.
Am J Physiol. 1989 Feb;256(2 Pt 1):C219-25. doi: 10.1152/ajpcell.1989.256.2.C219.
The formation of unactivated and activated glucocorticoid receptor complexes was studied in intact, isolated, perfused rat hearts in the presence of [3H]triamcinolone acetonide. Receptor activation, as quantified by the DNA-cellulose-binding assay, began to increase within 30 s of perfusion and reached a final steady-state level (t 1/2 = 4.6 min) with 46% of the steroid-receptor complexes bound to DNA-cellulose. With the use of a linear potassium phosphate (KP) gradient (5-400 mM), unactivated receptors eluted from DEAE-cellulose anion exchange columns at approximately 250 mM KP. Two activated receptor forms appeared, which eluted either in the wash fraction (binder IB) or between 50 and 100 mM KP (binder II) and occurred with half times of 1.3 and 2.7 min, respectively. Postperfusion cytosol preparation did not markedly influence the results as receptor binding was reduced by 10% or less when a 100-fold excess of unlabeled triamcinolone acetonide was included in the homogenizing buffer. We conclude from these results that glucocorticoids are able to exert a direct effect on the heart through binding to their own receptor in the absence of endogenous hormones. The time dependency of receptor activation supports a physiological role for this process. However, activation rates, determined from conformational changes associated with altered DEAE-cellulose elution profiles and appearance of activated receptor forms, occur earlier and may not be coordinated with the rate of activation as quantified by DNA-cellulose binding.
在存在[3H]曲安奈德的情况下，对完整、分离、灌注的大鼠心脏中未活化和活化的糖皮质激素受体复合物的形成进行了研究。通过DNA-纤维素结合测定法量化的受体活化在灌注后30秒内开始增加，并达到最终稳态水平（t 1/2 = 4.6分钟），其中46%的类固醇-受体复合物与DNA-纤维素结合。使用线性磷酸钾（KP）梯度（5-400 mM）时，未活化的受体在约250 mM KP处从DEAE-纤维素阴离子交换柱上洗脱。出现了两种活化的受体形式，一种在洗脱部分（结合物IB）中洗脱，另一种在50至100 mM KP之间洗脱（结合物II），其半衰期分别为1.3分钟和2.7分钟。灌注后胞质溶胶制备对结果没有明显影响，因为当在匀浆缓冲液中加入100倍过量的未标记曲安奈德时，受体结合减少了10%或更少。我们从这些结果得出结论，在没有内源性激素的情况下，糖皮质激素能够通过与其自身受体结合对心脏发挥直接作用。受体活化的时间依赖性支持了这一过程的生理作用。然而，由与DEAE-纤维素洗脱谱改变和活化受体形式出现相关的构象变化确定的活化速率更早发生，并且可能与通过DNA-纤维素结合量化的活化速率不协调。
