Stanislaus D, Janovick J A, Jennes L, Kaiser U B, Chin W W, Conn P M
Oregon Health Sciences University, Portland 97201.
Endocrinology. 1994 Nov;135(5):2220-7. doi: 10.1210/endo.135.5.7956945.
Four cell lines, stably transfected with rat GnRH receptor complementary DNA, have been prepared from the lactotropic GH3 cell line. All four lines (as well as the parent line and a line transfected with the vector DNA) show extensive rosettes of circular polyribosomes, characteristic of high protein synthetic activity, although secretory granules are virtually absent; the rough endoplasmic reticulum (rER) cisternae were short and straight. Instances were observed in which the ER reaches to the plasma membrane, suggesting a possible nongranular secretory route. All four lines (but not the parent or a control transfected line) expressed GnRH receptors that were down-regulated (1-5 h, depending on the cell line) after exposure to 10 nM GnRH; receptors then recovered (2-7 h). This pattern is reminiscent of the GnRH receptor in the primary gonadotrope cell cultures. All cell lines released PRL (4-96 h) in response to a GnRH agonist (D-tBuSer6-des-Gly10-Pro9-ethylamide-GnRH), an event that was inhibited by all three major classes of Ca+2 ion channel antagonists (methoxyverapamil, 1,4-dihydropyridines, and diltiazem); in contrast, GnRH-stimulated LH release from pituitary-derived primary cultures is only inhibited by methoxyverapamil. One line became refractory to GnRH analog stimulation after 24 h, although the other three released PRL vigorously up to the longest time point examined (96 h). All four lines responded substantially more robustly to 1 microgram/ml Buserelin than to 1 microgram/ml TRH. All four lines produced inositol phosphate metabolites and released immunoassayable cAMP (24 h) in response to treatment with Buserelin. These cell lines are good models for understanding the mechanisms by which the GnRH receptor is coupled to second messenger systems and for comparing these mechanisms with TRH-receptor coupling in the same cell.
已从促乳素分泌型GH3细胞系制备出4种稳定转染大鼠促性腺激素释放激素（GnRH）受体互补DNA的细胞系。所有这4种细胞系（以及亲代细胞系和转染载体DNA的细胞系）均显示出大量环状多聚核糖体玫瑰花结，这是高蛋白合成活性的特征，尽管几乎没有分泌颗粒；粗面内质网（rER）池短而直。观察到内质网延伸至质膜的情况，提示可能存在非颗粒性分泌途径。所有这4种细胞系（但亲代细胞系或对照转染细胞系未出现此情况）均表达GnRH受体，在暴露于10 nM GnRH后，该受体会下调（1 - 5小时，取决于细胞系），随后受体恢复（2 - 7小时）。这种模式类似于原代促性腺激素细胞培养中的GnRH受体。所有细胞系在GnRH激动剂（D - tBuSer6 - des - Gly10 - Pro9 - 乙酰胺 - GnRH）作用下均释放催乳素（PRL，4 - 96小时），这一事件受到所有三大类Ca + 2离子通道拮抗剂（甲氧基维拉帕米、1,4 - 二氢吡啶和地尔硫䓬）的抑制；相比之下，GnRH刺激垂体来源的原代培养物释放促黄体生成素（LH）仅受甲氧基维拉帕米抑制。其中一个细胞系在24小时后对GnRH类似物刺激产生耐受，尽管其他三个细胞系在最长检测时间点（96小时）仍能强烈释放PRL。所有这4种细胞系对1微克/毫升布舍瑞林的反应比对1微克/毫升促甲状腺激素释放激素（TRH）的反应要强得多。所有这4种细胞系在布舍瑞林处理后均产生肌醇磷酸代谢物并释放可通过免疫测定的环磷酸腺苷（cAMP，24小时）。这些细胞系是理解GnRH受体与第二信使系统偶联机制以及在同一细胞中将这些机制与TRH受体偶联进行比较的良好模型。
