Naor Z, Katikineni M, Loumaye E, Vela A G, Dufau M L, Catt K J
Mol Cell Endocrinol. 1982 Jul;27(2):213-20. doi: 10.1016/0303-7207(82)90110-1.
Cultured rat anterior pituitary cells were continuously perfused with medium 199, and exposed to short (5 min), intermediate (30 min), or long (6 h) pulses of a maximally effective concentration of gonadotropin-releasing hormone (GnRH). Assay of the effluent by radioimmunoassay and interstitial-cell bioassay revealed a biphasic response to GnRH, and indicated that 3 pools of luteinizing hormone (LH) are present in the gonadotroph. A rapidly releasable peak of bioactive LH comprising about 2% of the total cellular LH was mobilized within 1 min of GnRH addition, lasted for 3-4 min, and was independent of the duration of stimulation. The second, larger pool of bioactive LH varied from 15 to 50% of the total LH as the duration of exposure to GnRH was increased from 5 min to 6 h. A third LH pool comprising up to 50% of the total LH could be mobilized by 50 mM potassium but not by continuous GnRH treatment, due to refractoriness of the cells to prolonged stimulation by the decapeptide. In contrast, repeated pulses of GnRH evoked a series of biphasic LH peaks with profiles similar to that observed during a single response to GnRH, indicating that continuous exposure to GnRH is necessary for densensitization. Release of LH from the perfused cells was calcium-dependent, and the bio-immuno ratio of the first and second pools of LH was similar. The in vitro secretion profile of cultured rat cells is comparable with the early and late phases of LH release observed in GnRH-infused man, but occurs much more rapidly, and demonstrates heterogeneity of the LH release process at the level of the gonadotroph. The superfusion technique provides a powerful tool to further investigate the bioactivity of GnRH and its analogs for use in fertility control.
用199培养基连续灌注培养的大鼠垂体前叶细胞，并使其暴露于最大有效浓度的促性腺激素释放激素（GnRH）的短脉冲（5分钟）、中脉冲（30分钟）或长脉冲（6小时）。通过放射免疫测定法和间质细胞生物测定法对流出物进行分析，结果显示对GnRH呈双相反应，并表明促性腺细胞中存在3个促黄体生成素（LH）池。在添加GnRH后1分钟内，动员了一个快速释放的生物活性LH峰，约占细胞总LH的2%，持续3 - 4分钟，且与刺激持续时间无关。随着GnRH暴露时间从5分钟增加到6小时，第二个更大的生物活性LH池占总LH的15%至50%。第三个LH池占总LH的比例高达50%，可通过50 mM钾动员，但不能通过持续的GnRH处理动员，这是由于细胞对十肽的长期刺激产生了不应性。相比之下，重复的GnRH脉冲诱发了一系列双相LH峰，其轮廓与单次对GnRH反应时观察到的相似，表明持续暴露于GnRH对于脱敏是必要的。灌注细胞中LH的释放是钙依赖性的，并且第一和第二LH池的生物免疫比率相似。培养的大鼠细胞的体外分泌模式与在注入GnRH的男性中观察到的LH释放的早期和晚期阶段相当，但发生得更快，并且在促性腺细胞水平上证明了LH释放过程的异质性。超灌注技术为进一步研究GnRH及其类似物在生育控制中的生物活性提供了一个强大的工具。
