Sömjen D, Kohen F, Lieberherr M
Endocrine Unit, Tel Aviv Medical Center, Israel.
J Cell Biochem. 1997 Apr;65(1):53-66. doi: 10.1002/(sici)1097-4644(199704)65:1<53::aid-jcb6>3.0.co;2-y.
We investigated the early effects of the anti-idiotypic antibody (clone 1D5), which recognized the estrogen receptor (ER), on cytosolic free calcium concentration ([Ca2+]i) and its long term effects on creatine kinase (CK) specific activity in female human and rat osteoblasts. These actions were compared to the known membrane and genomic effects of 17 beta estradiol (E2). Like E2, clone 1D5 increased within 5 s [Ca2+]i in both cell types by two mechanisms: 1) Ca2+ influx through voltage-gated Ca2+ channels as shown by using EGTA a chelator of extracellular Ca2+, and nifedipine, a Ca2+ channel blocker; 2) Ca2+ mobilization from the endoplasmic reticulum as shown by using phospholipase C inhibitors, such as neomycin and U-73122, which involved a Pertussis toxin-sensitive G-protein. Clone 1D5 and E2 stimulated CK specific activity in human and rat osteoblasts with ten fold higher concentrations than those needed for the membrane effects (0.1 microgram/ml and 10 pM, respectively). Both effects were gender-specific since testosterone and 5 alpha-dihydotesterone were uneffective. Tamoxifen and Raloxifene, two estrogen nuclear antagonists, inhibited CK response to 1D5 and E2 and Ca2+ response to 1D5, but not Ca2+ response to E2. By contrast, (Fab')2 dimer, a proteolytic fragment of 1D5 with antagonist properties, inhibited both membrane and genomic effects of 1D5 and E2. In conclusion, these results imply that clone 1D5 has an estrogen like activity both at the membrane and nuclear levels in female human and rat osteoblasts. 1D5 must therefore interact with membrane binding sites, penetrate the cells, and reach the nuclear receptors by an as yet uncharacterized mechanism.
我们研究了识别雌激素受体（ER）的抗独特型抗体（克隆1D5）对女性人类和大鼠成骨细胞胞质游离钙浓度（[Ca2+]i）的早期影响及其对肌酸激酶（CK）比活性的长期影响。将这些作用与已知的17β-雌二醇（E2）的膜效应和基因组效应进行了比较。与E2一样，克隆1D5在5秒内通过两种机制增加了两种细胞类型中的[Ca2+]i：1）通过使用细胞外Ca2+螯合剂乙二醇双四乙酸（EGTA）和Ca2+通道阻滞剂硝苯地平表明，Ca2+通过电压门控Ca2+通道流入；2）通过使用磷脂酶C抑制剂如新霉素和U-73122表明，Ca2+从内质网中动员出来，这涉及一种百日咳毒素敏感的G蛋白。克隆1D5和E2刺激人类和大鼠成骨细胞中的CK比活性，其浓度比膜效应所需浓度高十倍（分别为0.1微克/毫升和10皮摩尔）。两种效应均具有性别特异性，因为睾酮和5α-二氢睾酮无效。两种雌激素核拮抗剂他莫昔芬和雷洛昔芬抑制了CK对1D5和E2的反应以及Ca2+对1D5的反应，但不抑制Ca2+对E2的反应。相比之下，具有拮抗剂特性的1D5蛋白水解片段（Fab'）2二聚体抑制了1D5和E2的膜效应和基因组效应。总之，这些结果表明克隆1D5在女性人类和大鼠成骨细胞的膜水平和核水平均具有雌激素样活性。因此，1D5必须与膜结合位点相互作用，穿透细胞，并通过一种尚未明确的机制到达核受体。
