The L beta T2 clonal gonadotrope: a model for single cell studies of endocrine cell secretion.
The L beta T2 clonal gonadotrope: a model for single cell studies of endocrine cell secretion.
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DOI：
10.1210/endo.137.7.8770922
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发表时间：
1996-07
期刊：
影响因子：
4.8
通讯作者：
Paul Thomas;P. Mellon;J. Turgeon;D. W. Waring
中科院分区：
文献类型：
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作者：
Paul Thomas;P. Mellon;J. Turgeon;D. W. Waring
我们已经使用了单一的促性腺激素从新衍生的线，LbetaT 2，研究调节钙信号和胞吐的类固醇激素环境。该细胞系是通过转基因小鼠的靶向肿瘤发生而衍生的，最近已被证明可以响应GnRH而分泌LH。我们已经表征了GnRH和膜去极化对单个LbetaT 2细胞中胞吐和细胞内[Ca 2 +]（[Ca 2 +]i）的影响。GnRH（1-100 nM）诱发[Ca 2 +]i和分泌的浓度依赖性增加，通过使用全细胞穿孔贴片技术测量质膜电容（Cm）进行监测，这些变化的程度取决于类固醇激素背景。GnRH处理的细胞培养在培养基中含有炭处理的FBS（ct-FBS）显示较小的变化[Ca 2 +]i比细胞培养在未经处理的FBS。然而，当雌二醇（E2）和地塞米松（Dex）添加到ct-FBS培养基（E2/Dex-ct-FBS）时，GnRH刺激的[Ca 2 +]i升高几乎增加了2倍。此外，E2/Dex-ct-FBS培养的细胞中的分泌速率大于ct-FBS或FBS培养的细胞。用E2/Dex-ct-FBS观察到的分泌反应增加似乎是由于GnRH刺激的峰值[Ca 2 +]i增加和胞吐作用的Ca 2+依赖性的敏感性增加。与GnRH诱发的反应相反，在ct-FBS中培养的细胞中由去极化引起的[Ca 2 +]i的增加大于E2/Dex-ct-FBS;然而，两组的分泌率没有差异。同样，类固醇治疗对去极化诱发的胞吐的Ca 2+依赖性没有明显的影响。总之，这些结果1）清楚地证明了该细胞系用于激动剂和去极化诱发的分泌的单细胞研究的实用性; 2）揭示了类固醇激素背景对L β T2细胞具有深远的影响，包括对刺激诱导的钙动员和对胞吐的表观Ca 2+敏感性的影响;和3）显示类固醇激素对Ca 2+敏感性的作用的表达依赖于GnRH对受体的占据。
We have used single gonadotropes from the newly derived line, LbetaT2, to investigate the modulation of Ca2+ signaling and exocytosis by the steroid hormone environment. This cell line, derived by targeted oncogenesis in transgenic mice, has recently been shown to secrete LH in response to GnRH. We have characterized the effects of both GnRH and membrane depolarization on exocytosis and intracellular [Ca2+] ([Ca2+]i) in individual LbetaT2 cells. GnRH (1-100 nM) evoked concentration-dependent increases in [Ca2+]i and secretion, as monitored by measurement of plasma membrane capacitance (Cm) using the whole-cell perforated-patch technique, and the extent of these changes were dependent upon steroid hormone background. GnRH treatment of cells cultured in medium containing charcoal-treated FBS (ct-FBS) showed smaller changes in [Ca2+]i than cells cultured in untreated FBS. However, when estradiol (E2) and dexamethasone (Dex) were added to the ct-FBS medium (E2/Dex-ct-FBS), the elevations in [Ca2+]i stimulated by GnRH increased almost 2-fold. Additionally, the rates of secretion in the E2/Dex-ct-FBS-cultured cells were greater than in either ct-FBS- or FBS-cultured cells. The increase in secretory response observed with E2/Dex-ct-FBS appeared to be due to both an increase in the peak [Ca2+]i stimulated by GnRH and a shift toward increased sensitivity of the Ca2+ dependency of exocytosis. In contrast to GnRH-evoked responses, the increases in [Ca2+]i elicited by depolarization were greater in cells cultured in ct-FBS than in E2/Dex-ct-FBS; however, the secretory rates were no different in the two groups. Likewise, there was no apparent effect of steroid treatment on the Ca2+ dependency of depolarization-evoked exocytosis. In summary, these results 1) clearly demonstrate the utility of this cell line for single-cell studies of both agonist- and depolarization-evoked secretion; 2) reveal that steroid hormone background has profound effects on LbetaT2 cells, both on stimulus-induced calcium mobilization and on the apparent Ca2+ -sensitivity of exocytosis; and 3) show that expression of the steroid hormone effect on Ca2+ -sensitivity is dependent upon receptor occupation by GnRH.