Gershengorn M C, Geras E, Purrello V S, Rebecchi M J
J Biol Chem. 1984 Sep 10;259(17):10675-81.
Thyrotropin-releasing hormone (TRH) stimulation of prolactin secretion from GH3 cells, cloned rat pituitary tumor cells, is associated with 1) hydrolysis of phosphatidylinositol 4,5-bisphosphate to yield inositol trisphosphate (InsP3) and 2) elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i), caused in part by mobilization of cellular calcium. We demonstrate, in intact cells, that TRH mobilizes calcium and, in permeabilized cells, that InsP3 releases calcium from a nonmitochondrial pool(s). In intact cells, TRH caused a loss of 16 +/- 2.7% of cell-associated 45Ca which was not inhibited by depleting the mitochondrial calcium pool with uncoupling agents. Similarly, TRH caused an elevation of [Ca2+]i from 127 +/- 6.3 nM to 375 +/- 54 nM, as monitored with Quin 2, which was not inhibited by depleting mitochondrial calcium. Saponin-permeabilized cells accumulated Ca2+ in an ATP-dependent manner into a nonmitochondrial pool, which exhibited a high affinity for Ca2+ and a small capacity, and into a mitochondrial pool which had a lower affinity for Ca2+ but was not saturated under the conditions tested. Permeabilized cells buffered free Ca2+ to 129 +/- 9.2 nM when incubated in a cytosol-like solution initially containing 200 to 1000 nM free Ca2+. InsP3, but not other inositol sugars, released calcium from the nonmitochondrial pool(s); half-maximal effect occurred at approximately 1 microM InsP3. Ca2+ release was followed by reuptake into a nonmitochondrial pool(s). These data suggest that InsP3 serves as an intracellular mediator (or second messenger) of TRH action to mobilize calcium from a nonmitochondrial pool(s) leading to an elevation of [Ca2+]i and then to prolactin secretion.
促甲状腺激素释放激素（TRH）刺激克隆的大鼠垂体肿瘤细胞GH3细胞分泌催乳素，这与以下过程相关：1）磷脂酰肌醇4,5 - 二磷酸水解生成肌醇三磷酸（InsP3）；2）细胞质游离钙离子浓度[Ca2+]i升高，这部分是由细胞内钙的动员引起的。我们在完整细胞中证明TRH能动员钙，在通透细胞中证明InsP3能从非线粒体钙库释放钙。在完整细胞中，TRH导致细胞相关的45Ca损失16±2.7%，用解偶联剂耗尽线粒体钙库并不能抑制这一过程。同样，用喹啉2监测发现，TRH使[Ca2+]i从127±6.3 nM升高到375±54 nM，耗尽线粒体钙也不能抑制这一升高。皂角苷通透的细胞以ATP依赖的方式将Ca2+积累到一个对Ca2+具有高亲和力且容量较小的非线粒体钙库以及一个对Ca2+亲和力较低但在测试条件下不饱和的线粒体钙库中。当在最初含有200至1000 nM游离Ca2+的类似细胞质的溶液中孵育时，通透细胞将游离Ca2+缓冲到129±9.2 nM。InsP3而非其他肌醇糖能从非线粒体钙库释放钙；半最大效应发生在约1 μM InsP3时。Ca2+释放后会重新摄取到非线粒体钙库中。这些数据表明InsP3作为TRH作用的细胞内介质（或第二信使），从非线粒体钙库动员钙，导致[Ca2+]i升高，进而引起催乳素分泌。
