Ramey J W, Krummen L A, Wilfinger W W, Highsmith R F, Baldwin D M
Endocrinology. 1987 Apr;120(4):1514-20. doi: 10.1210/endo-120-4-1514.
The purpose of this study was to investigate the effects of lowering the extracellular calcium concentration on GnRH-stimulated LH glycosylation and LH translation, as measured by the incorporation of [3H]glucosamine (3H-Gln) and [35S]methionine (35S-Met) into immunoprecipitable LH. Cultured anterior pituitary cells, previously exposed to estradiol (5 X 10(-10) M) to maximize precursor incorporation were incubated for 4 h in normal calcium (2.5 mM) or low calcium medium (less than 15 microM) containing radiolabeled precursors with or without 1 nM GnRH. In the presence of normal calcium, GnRH significantly increased 3H-Gln-labeled LH in the medium (278%) and cells (290%), as well as total (cells plus medium) 3H- Gln LH (280%) compared to the control value (no GnRH). GnRH also significantly increased the 35S-Met LH released into the medium (164%) and total 35S-Met LH (186%) over control values. Depletion of extracellular calcium completely inhibited GnRH-stimulated 3H-Gln LH and 35S-Met LH production. Total immunoreactive LH (iLH), as measured by RIA, was also increased significantly by GnRH treatment in the presence of calcium, but this response was prevented by removal of calcium from the medium. Lowering extracellular calcium had no effect on cellular uptake or incorporation of 3H-Gln or 35S-Met into total trichloroacetic acid-precipitable protein. Approximately 80% of newly synthesized LH was released into the medium in all treatment groups independent of whether calcium or GnRH was present. The specific activity (disintegrations per min/microgram iLH) of radiolabeled LH released into the medium was significantly reduced by treatment with GnRH due to the large amount of unlabeled iLH released into the medium. However, when the cells were incubated in low calcium, the SA of 3H-Gln LH and 35S-Met LH in the medium was unaltered by GnRH, whereas GnRH-stimulated iLH release was inhibited. We conclude that GnRH stimulation of LH glycosylation and LH apoprotein synthesis involves extracellular calcium-dependent events, and the release of newly synthesized LH is closely coupled to LH biosynthesis and is less dependent on extracellular calcium, whereas the GnRH-stimulated release of previously synthesized, stored LH is dependent on extracellular calcium.
本研究的目的是通过检测[3H]葡糖胺（3H-Gln）和[35S]甲硫氨酸（35S-Met）掺入可免疫沉淀的促黄体生成素（LH）的情况，来研究降低细胞外钙浓度对促性腺激素释放激素（GnRH）刺激的LH糖基化和LH翻译的影响。将先前暴露于雌二醇（5×10⁻¹⁰ M）以最大化前体掺入的培养垂体前叶细胞，在含有放射性标记前体且添加或不添加1 nM GnRH的正常钙（2.5 mM）或低钙培养基（小于15 μM）中孵育4小时。在正常钙存在的情况下，与对照值（无GnRH）相比，GnRH显著增加了培养基中（278%）和细胞中（290%）3H-Gln标记的LH，以及总的（细胞加培养基）3H-Gln LH（280%）。GnRH还使释放到培养基中的35S-Met LH（164%）和总的35S-Met LH（186%）显著高于对照值。细胞外钙的耗尽完全抑制了GnRH刺激的3H-Gln LH和35S-Met LH的产生。通过放射免疫分析（RIA）测量的总免疫反应性LH（iLH），在有钙存在的情况下，GnRH处理也使其显著增加，但培养基中钙的去除阻止了这种反应。降低细胞外钙对细胞摄取或3H-Gln或35S-Met掺入总的三氯乙酸可沉淀蛋白没有影响。在所有处理组中，无论是否存在钙或GnRH，大约80%新合成的LH释放到培养基中。由于大量未标记的iLH释放到培养基中，用GnRH处理显著降低了释放到培养基中的放射性标记LH的比活性（每分钟衰变数/微克iLH）。然而，当细胞在低钙条件下孵育时，培养基中3H-Gln LH和35S-Met LH的比活性不受GnRH影响，而GnRH刺激的iLH释放受到抑制。我们得出结论，GnRH刺激的LH糖基化和LH载脂蛋白合成涉及细胞外钙依赖性事件，新合成的LH的释放与LH生物合成密切相关且对细胞外钙的依赖性较小，而GnRH刺激的先前合成、储存的LH的释放则依赖于细胞外钙。
