Vedder H, Weiss I, Holsboer F, Reul J M
Max Planck Institute of Psychiatry, Department of Neuroendocrinology, Munich, FRG.
Brain Res. 1993 Mar 5;605(1):18-24. doi: 10.1016/0006-8993(93)91351-r.
Glucocorticoid and mineralocorticoid binding sites were characterized in cell cultures derived from neocortical and hippocampal brain tissue from fetal (E18) rats. Specific and saturable binding was detected in living cells with a sensitive whole cell binding method using [3H]dexamethasone ([3H]DEX) and [3H]aldosterone ([3H]ALDO) (in the presence of RU 28362, a selective glucocorticoid receptor (GR) agonist) as ligands for the measurement of glucocorticoid and mineralocorticoid receptors (MRs), respectively. Specific corticosteroid binding was demonstrated as early as day 4 in culture in neocortical cells, with a time-dependent increase in binding sites during further culturing time. At 7-9 days in vitro, Scatchard analysis of [3H]DEX binding revealed a maximum binding capacity (Bmax) of 83.4 +/- 5.0 fmol/mg protein and a binding affinity (Kd) of 3.6 +/- 0.4 nM in neocortical brain cells. Competition binding studies with [3H]DEX demonstrated a glucocorticoid specificity of receptor sites (relative binding affinity: RU 28362 = DEX > PROG > ALDO). Similar binding characteristics were demonstrated for GRs in cultures derived from fetal hippocampal tissue (Bmax 49.1 +/- 5.8 fmol/mg protein, Kd 3.5 +/- 0.2 nM). Analysis of MRs with [3H]ALDO (+RU 28362) revealed specific and saturable binding in hippocampal cultures, with a Bmax of 8.0 +/- 0.5 fmol/mg protein and a Kd of 0.2 +/- 0.1 nM. Competition studies with [3H]ALDO showed a mineralocorticoid-like pattern of receptor binding (relative binding affinity: CORT = ALDO > PROG > DEX). In addition, small numbers of MRs were detectable in cortex-derived cultures (Bmax: 3.7 +/- 0.8 fmol/mg protein, Kd: 0.3 +/- 0.2 nM).(ABSTRACT TRUNCATED AT 250 WORDS)
在源自胎鼠（E18）新皮质和海马脑组织的细胞培养物中，对糖皮质激素和盐皮质激素结合位点进行了表征。使用[3H]地塞米松（[3H]DEX）和[3H]醛固酮（[3H]ALDO）（在选择性糖皮质激素受体（GR）激动剂RU 28362存在的情况下）作为配体，分别用于测量糖皮质激素受体和盐皮质激素受体（MRs），通过灵敏的全细胞结合方法在活细胞中检测到特异性和可饱和结合。新皮质细胞培养至第4天时就证明了特异性皮质类固醇结合，在进一步培养过程中结合位点呈时间依赖性增加。在体外培养7 - 9天时，对[3H]DEX结合的Scatchard分析显示，新皮质脑细胞的最大结合容量（Bmax）为83.4±5.0 fmol/mg蛋白质，结合亲和力（Kd）为3.6±0.4 nM。用[3H]DEX进行的竞争结合研究证明了受体位点的糖皮质激素特异性（相对结合亲和力：RU 28362 = DEX > PROG > ALDO）。源自胎鼠海马组织的培养物中的GR也表现出类似的结合特性（Bmax 49.1±5.8 fmol/mg蛋白质，Kd 3.5±0.2 nM）。用[3H]ALDO（+RU 28362）对MRs的分析显示，海马培养物中存在特异性和可饱和结合，Bmax为8.0±0.5 fmol/mg蛋白质，Kd为0.2±0.1 nM。用[3H]ALDO进行的竞争研究显示出类似盐皮质激素的受体结合模式（相对结合亲和力：CORT = ALDO > PROG > DEX）。此外，在源自皮质的培养物中可检测到少量MRs（Bmax：3.7±0.8 fmol/mg蛋白质，Kd：0.3±0.2 nM）。（摘要截断于250字）
