Aron D C, Roos B A
Endocrinology. 1986 Jan;118(1):218-22. doi: 10.1210/endo-118-1-218.
We have established a system, the CA77 rat medullary thyroid carcinoma cell line, for studying the products of somatostatin (SS) gene expression. Based on the amino acid sequence of proSS, we developed a RIA for the amino terminus of proSS (proSS-NTP) and demonstrated in acidic cell extracts two major proSS-NTP-containing species of 8000 and 4000 daltons. Studies were then performed on species secreted into culture medium. Serial dilutions of culture medium showed tracer displacement curves parallel to serial dilutions of synthetic proSS-NTP standard. Analysis by gel filtration chromatography of 24-h culture medium showed the major proSS-NTP-containing species to have an estimated mol wt of 8000 daltons. No 4000-dalton species was observed. The acute effects of calcium and glucagon, known secretagogues of SS, on secretion of immunoreactive (i) proSS-NTP were investigated in 3-h experiments. Basal (0.5 mM calcium) secretory rates (mean +/- SE) of iproSS-NTP and iSS were 1.29 +/- 0.36 and 7.38 +/- 1.51 ng/mg acid-extractable protein, respectively. High calcium (3 mM) stimulated iproSS-NTP and iSS secretion 302 +/- 100% and 363 +/- 105%, respectively. High calcium plus 10(-6) M glucagon also stimulated secretion of iproSS-NTP and iSS in a coordinate fashion. Analyses by gel filtration chromatography of 3-h culture medium revealed that high calcium markedly increased the 8000-dalton proSS-NTP-containing species. No 4000-dalton species was observed. The absence of 4000-dalton proSS-NTP species in 24-h culture medium, the lack of degradation of 4000-dalton proSS-NTP (recovered from CA77 cell extracts) added to tissue culture medium, and the selective secretion of the 8000-dalton proSS-NTP species under both basal and stimulated conditions coordinate with the secretion of SS indicate that the 4000-dalton proSS-NTP-containing species is not secreted.
我们建立了一个系统——CA77大鼠甲状腺髓样癌细胞系，用于研究生长抑素（SS）基因表达的产物。基于前体SS的氨基酸序列，我们开发了一种针对前体SS氨基末端（proSS-NTP）的放射免疫分析方法，并在酸性细胞提取物中证实了两种主要的含proSS-NTP的物质，分子量分别为8000和4000道尔顿。随后对分泌到培养基中的物质进行了研究。培养基的系列稀释显示示踪剂置换曲线与合成proSS-NTP标准品的系列稀释曲线平行。对24小时培养基进行凝胶过滤色谱分析表明，主要的含proSS-NTP的物质估计分子量为8000道尔顿。未观察到4000道尔顿的物质。在3小时的实验中，研究了已知的SS促分泌剂钙和胰高血糖素对免疫反应性（i）proSS-NTP分泌的急性影响。iproSS-NTP和iSS的基础（0.5 mM钙）分泌率（平均值±标准误）分别为1.29±0.36和7.38±1.51 ng/mg酸可提取蛋白。高钙（3 mM）分别刺激iproSS-NTP和iSS分泌302±100%和363±105%。高钙加10^(-6) M胰高血糖素也以协同方式刺激iproSS-NTP和iSS的分泌。对3小时培养基进行凝胶过滤色谱分析表明，高钙显著增加了含8000道尔顿proSS-NTP的物质。未观察到4000道尔顿的物质。24小时培养基中不存在4000道尔顿的proSS-NTP物质，添加到组织培养基中的4000道尔顿proSS-NTP（从CA77细胞提取物中回收）未降解，以及在基础和刺激条件下8000道尔顿proSS-NTP物质的选择性分泌与SS的分泌协同，表明含4000道尔顿proSS-NTP的物质不被分泌。
