Orti E, Coirini H, De Nicola A F
Neuroendocrinology. 1985 Mar;40(3):225-31. doi: 10.1159/000124134.
We have examined the spinal cord for the presence of glucocorticoid-binding sites. For this purpose, cytosol from the spinal cord of adrenalectomized rats was incubated with (3H)-dexamethasone. Maximal binding was obtained after 20 h of incubation at 0 degree C in the presence of 20 mM molybdate, whereas at 20 degrees C the maximum was at 2 h. Using a range of (3H)-dexamethasone concentrations (0.2-30 nM), low capacity (161 +/- 23 fmol/mg protein) and high affinity (Kd 3.2 +/- 0.3 nM) sites were measured. Binding sites decreased by 25% and Kd increased 2.5-fold after incubation with a pure glucocorticoid (RU 26988). Relative binding affinities of several competitors of 10 nM (3H)-dexamethasone were: triamcinolone acetonide 108, dexamethasone 100, RU 26988 54, corticosterone 18, progesterone 17, aldosterone 7, estradiol and testosterone less than 1. Sedimentation coefficients in glycerol gradients containing molybdate were in the range of those published for glucocorticoid receptors (9.6-9.8 S). Binding of (3H)-dexamethasone was decreased by omitting a SH-protective agent from the buffer or by addition of SH-blocking reagents such as N-ethylmaleimide and p-chloromercuribenzoate. Using rats of different ages, it was found that binding sites were much lower in spinal cord from 2- to 8-day-old rats than in rats of 13-20 days and adults. Regional distribution studies using cytosol from spinal cords dissected between vertebrae C1-C2, C3-C7, T1-T8, T9-L3 and L4-L6 revealed that binding sites were higher in regions containing the cervical (C3-C7) and lumbar (T9-L3) enlargements, with respect to L4-L6.(ABSTRACT TRUNCATED AT 250 WORDS)
我们检测了脊髓中糖皮质激素结合位点的存在情况。为此，将肾上腺切除大鼠脊髓的胞质溶胶与（3H）-地塞米松一起孵育。在0℃、20 mM钼酸盐存在的条件下孵育20小时后可获得最大结合量，而在20℃时，2小时达到最大值。使用一系列（3H）-地塞米松浓度（0.2 - 30 nM），测得低容量（161±23 fmol/mg蛋白质）和高亲和力（Kd 3.2±0.3 nM）的位点。与纯糖皮质激素（RU 26988）孵育后，结合位点减少25%，Kd增加2.5倍。几种10 nM（3H）-地塞米松竞争剂的相对结合亲和力为：曲安奈德108、地塞米松100、RU 26988 54、皮质酮18、孕酮17、醛固酮7、雌二醇和睾酮小于1。含钼酸盐的甘油梯度中的沉降系数在已发表的糖皮质激素受体的范围内（9.6 - 9.8 S）。通过从缓冲液中省略SH保护剂或添加SH阻断剂如N-乙基马来酰亚胺和对氯汞苯甲酸，（3H）-地塞米松的结合减少。使用不同年龄的大鼠发现，2至8日龄大鼠脊髓中的结合位点比13至20日龄大鼠和成年大鼠的低得多。使用从C1 - C2、C3 - C7、T1 - T8、T9 - L3和L4 - L6椎骨间解剖的脊髓胞质溶胶进行的区域分布研究表明，相对于L4 - L6，含有颈膨大（C3 - C7）和腰膨大（T9 - L3）的区域结合位点更高。（摘要截于250字）
