From the Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523 and.
Department of Pharmacology and Toxicology, Center for Molecular Signaling (PZMS), Saarland University School of Medicine, 66421 Homburg, Germany.
J Biol Chem. 2018 Oct 12;293(41):16028-16042. doi: 10.1074/jbc.RA118.001830. Epub 2018 Aug 28.
Gonadotropin-releasing hormone (GnRH) stimulation of its eponymous receptor on the surface of endocrine anterior pituitary gonadotrope cells (gonadotropes) initiates multiple signaling cascades that culminate in the secretion of luteinizing and follicle-stimulating hormones, which have critical roles in fertility and reproduction. Enhanced luteinizing hormone biosynthesis, a necessary event for ovulation, requires a signaling pathway characterized by calcium influx through L-type calcium channels and subsequent activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK). We previously reported that highly localized subplasmalemmal calcium microdomains produced by L-type calcium channels (calcium sparklets) play an essential part in GnRH-dependent ERK activation. Similar to calcium, reactive oxygen species (ROS) are ubiquitous intracellular signaling molecules whose subcellular localization determines their specificity. To investigate the potential influence of oxidant signaling in gonadotropes, here we examined the impact of ROS generation on L-type calcium channel function. Total internal reflection fluorescence (TIRF) microscopy revealed that GnRH induces spatially restricted sites of ROS generation in gonadotrope-derived αT3-1 cells. Furthermore, GnRH-dependent stimulation of L-type calcium channels required intracellular hydrogen peroxide signaling in these cells and in primary mouse gonadotropes. NADPH oxidase and mitochondrial ROS generation were each necessary for GnRH-mediated stimulation of L-type calcium channels. Congruently, GnRH increased oxidation within subplasmalemmal mitochondria, and L-type calcium channel activity correlated strongly with the presence of adjacent mitochondria. Collectively, our results provide compelling evidence that NADPH oxidase activity and mitochondria-derived hydrogen peroxide signaling play a fundamental role in GnRH-dependent stimulation of L-type calcium channels in anterior pituitary gonadotropes.
促性腺激素释放激素 (GnRH) 与其在内分泌前垂体促性腺细胞 (促性腺细胞) 表面的同源受体结合，启动多种信号级联反应，最终导致黄体生成素和卵泡刺激素的分泌，这些激素在生育和繁殖中起着至关重要的作用。增强黄体生成素生物合成是排卵所必需的事件，需要一种信号通路，其特征是通过 L 型钙通道内流和随后激活丝裂原活化蛋白激酶细胞外信号调节激酶 (ERK)。我们之前的研究报告表明，L 型钙通道产生的高度局域化的质膜下钙微区 (钙火花) 在 GnRH 依赖性 ERK 激活中起着至关重要的作用。与钙类似，活性氧 (ROS) 是普遍存在的细胞内信号分子，其亚细胞定位决定了它们的特异性。为了研究氧化应激信号在促性腺细胞中的潜在影响，我们在此研究了 ROS 生成对 L 型钙通道功能的影响。全内反射荧光 (TIRF) 显微镜显示，GnRH 诱导促性腺细胞衍生的αT3-1 细胞中 ROS 生成的空间限制部位。此外，在这些细胞和原代小鼠促性腺细胞中，GnRH 依赖性刺激 L 型钙通道需要细胞内过氧化氢信号。NADPH 氧化酶和线粒体 ROS 的产生对于 GnRH 介导的 L 型钙通道刺激都是必需的。一致地，GnRH 增加了质膜下线粒体中的氧化，并且 L 型钙通道活性与相邻线粒体的存在密切相关。总之，我们的研究结果提供了令人信服的证据，表明 NADPH 氧化酶活性和线粒体衍生的过氧化氢信号在 GnRH 依赖性刺激前垂体促性腺细胞中的 L 型钙通道中起着基本作用。
