Thomas P, Mellon P L, Turgeon J, Waring D W
Department of Human Physiology, University of California, Davis, 95616.
Endocrinology. 1996 Jul;137(7):2979-89. doi: 10.1210/endo.137.7.8770922.
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.
我们使用了新衍生的LbetaT2细胞系中的单个促性腺激素细胞，来研究类固醇激素环境对Ca2+信号传导和胞吐作用的调节。该细胞系是通过转基因小鼠中的靶向肿瘤发生而获得的，最近已证明其能对GnRH作出反应分泌LH。我们已经表征了GnRH和膜去极化对单个LbetaT2细胞中胞吐作用和细胞内[Ca2+]（[Ca2+]i）的影响。通过使用全细胞穿孔膜片钳技术测量质膜电容（Cm）来监测，GnRH（1-100 nM）引起[Ca2+]i和分泌的浓度依赖性增加，并且这些变化的程度取决于类固醇激素背景。在含有经活性炭处理的胎牛血清（ct-FBS）的培养基中培养的细胞，经GnRH处理后[Ca2+]i的变化比在未经处理的胎牛血清中培养的细胞小。然而，当将雌二醇（E2）和地塞米松（Dex）添加到ct-FBS培养基中（E2/Dex-ct-FBS）时，GnRH刺激引起的[Ca2+]i升高几乎增加了2倍。此外，在E2/Dex-ct-FBS培养基中培养的细胞的分泌速率高于在ct-FBS或FBS培养基中培养的细胞。在E2/Dex-ct-FBS中观察到的分泌反应增加似乎是由于GnRH刺激引起的[Ca2+]i峰值增加以及胞吐作用的Ca2+依赖性敏感性增加所致。与GnRH引起的反应相反，在ct-FBS中培养的细胞中，去极化引起的[Ca2+]i增加大于在E2/Dex-ct-FBS中培养的细胞；然而，两组的分泌速率没有差异。同样，类固醇处理对去极化引起的胞吐作用的Ca2+依赖性没有明显影响。总之，这些结果1）清楚地证明了该细胞系在激动剂和去极化引起的分泌的单细胞研究中的实用性；2）揭示了类固醇激素背景对LbetaT2细胞有深远影响，对刺激诱导的钙动员和胞吐作用的表观Ca2+敏感性均有影响；3）表明类固醇激素对Ca2+敏感性的影响表达取决于GnRH对受体的占据。
