Moses D F, González S, McEwen B S, De Nicola A F
Laboratorio de Bioquímica Neuroendocrina, Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina.
J Steroid Biochem Mol Biol. 1991 Jul;39(1):5-12. doi: 10.1016/0960-0760(91)90005-p.
We have used three experimental protocols to determine binding parameters for type I and type II glucocorticoid receptors in the spinal cord and hippocampus (HIPPO) from adrenalectomized rats. In protocol A, 0.5-20 nM [3H]dexamethasone (DEX) was incubated plus or minus a 1000-fold excess of unlabeled DEX, assuming binding to a two-site model. In protocol B, [3H]DEX competed with a single concentration of RU 28362 (500 nM), whereas in protocol C, we used a concentration of RU 28362 which varied in parallel to that of [3H]DEX, such as 500 x. Results of protocols A and C were qualitatively similar, in that: (1) Bmax for type I receptors favored the HIPPO, while the content of type II sites was comparable in the two tissues; (2) Kd was consistently lower for type I than for type II sites in both tissues; and (3) type II receptors from the spinal cord showed lower affinity than their homologous sites from HIPPO. This last result was also obtained when using protocol B. In contrast, protocol B yielded binding data indicating that type II sites were of similar or higher affinity than type I sites. Computer simulation of the binding protocols demonstrated that protocols A and C were the most theoretically reliable for estimating the Kd and Bmax of type I sites, and the predicted error was smaller for protocol C, in comparison with protocol B. We suggest that the noted differences in the Kd of type II receptors between the spinal cord and HIPPO could account for a difference in sensitivity of the two systems in the physiological adrenal hormone range.
我们采用了三种实验方案来测定肾上腺切除大鼠脊髓和海马体（HIPPO）中I型和II型糖皮质激素受体的结合参数。在方案A中，假定与双位点模型结合，将0.5 - 20 nM的[³H]地塞米松（DEX）与有无1000倍过量未标记DEX一起孵育。在方案B中，[³H]DEX与单一浓度（500 nM）的RU 28362竞争，而在方案C中，我们使用与[³H]DEX平行变化的RU 28362浓度，例如500倍。方案A和C的结果在质量上相似，即：（1）I型受体的Bmax在海马体中更占优势，而II型位点的含量在两个组织中相当；（2）在两个组织中，I型位点的Kd始终低于II型位点；（3）脊髓中的II型受体显示出比海马体中同源位点更低的亲和力。使用方案B时也得到了这一结果。相比之下，方案B产生的结合数据表明II型位点的亲和力与I型位点相似或更高。结合方案的计算机模拟表明，方案A和C在理论上对于估计I型位点的Kd和Bmax最可靠，并且与方案B相比，方案C的预测误差更小。我们认为，脊髓和海马体中II型受体Kd的显著差异可能解释了这两个系统在生理肾上腺激素范围内敏感性的差异。
