Walker A M, Farquhar M G
Endocrinology. 1980 Oct;107(4):1095-104. doi: 10.1210/endo-107-4-1095.
Pituitary cells maintained in monolayer culture for 48 h were used for double isotope labeling to study the release of newly synthesized vs. old PRL. The intracellular pathway taken by newly synthesized PRL was studied by autoradiography. For double labeling the cells were first incubated in a 14C-labeled amino acid (4 h) and then in a 3H-labeled amino acid (1 h), each followed by a 1-h chase. Total PRL (RIA) and radiolabeled PRL (immunoprecipitation) were determined in both cells and media. Thre rate of release of PRL (RIA) was stable throughout the experimental period. In unstimulated cells the ratio of 3H- to 14C-labeled PRL in the medium at the end of a 1-h incubation following the second chase was twice that in the cells at the beginning of this incubation, indicating that newly synthesized [3H]PRL was preferentially released. Stimulation with TRH resulted in increased release of older [14C]PRL. The earliest that radiolabeled (14C or 3H) PRL could be detected in the medium was 15--30 min after exposure of the cells to isotope. For autoradiography, cells were given a 5-min pulse of [3H]leucine, followed by chase periods of 30--180 min. Grain counts indicated that mammotrophs maintained in culture for 48 h transported and packaged PRL with the same time course as those preparations studied previously. Analysis of the distribution of total grains per cell in mammotrophs revealed the presence of several functional subpopulations with different numbers of total grains per cell, indicating that some cells were manufacturing and secreting PRL at a very fast rate. It is concluded that 1) preferential release of newly synthesized PRL is due to preferential discharge of newly synthesized granules, since release occurs at a time when labeled hormone is already in the granules; 2) there is no evidence that the established secretory pathway is bypassed, since transport, concentration, and granule formation occur; and 3) preferential release of newly synthesized PRL is the result of functional heterogeneity within the mammotroph population. Functional heterogeneity is illustrated by major differences in both synthetic rates and TRH responsiveness.
将单层培养48小时的垂体细胞用于双同位素标记，以研究新合成的与旧的催乳素（PRL）的释放情况。通过放射自显影研究新合成的PRL所采用的细胞内途径。进行双标记时，先将细胞在14C标记的氨基酸中孵育4小时，然后在3H标记的氨基酸中孵育1小时，每次孵育后都有1小时的追踪期。在细胞和培养基中分别测定总PRL（放射免疫分析，RIA）和放射性标记的PRL（免疫沉淀）。在整个实验期间，PRL（RIA）的释放速率保持稳定。在未受刺激的细胞中，第二次追踪后1小时孵育结束时培养基中3H标记的PRL与14C标记的PRL的比值是该孵育开始时细胞中该比值的两倍，这表明新合成的[3H]PRL优先释放。用促甲状腺激素释放激素（TRH）刺激导致旧的[14C]PRL释放增加。在细胞暴露于同位素后最早15 - 30分钟可在培养基中检测到放射性标记（14C或3H）的PRL。进行放射自显影时，给细胞5分钟的[3H]亮氨酸脉冲，随后是30 - 180分钟的追踪期。颗粒计数表明，培养48小时的泌乳细胞运输和包装PRL的时间进程与先前研究的那些制剂相同。对泌乳细胞中每个细胞的总颗粒分布分析显示存在几个功能性亚群，每个细胞的总颗粒数量不同，这表明一些细胞以非常快的速度制造和分泌PRL。得出的结论是：1）新合成的PRL优先释放是由于新合成颗粒的优先排出，因为释放发生在标记激素已存在于颗粒中的时候；2）没有证据表明既定的分泌途径被绕过，因为发生了运输、浓缩和颗粒形成；3）新合成的PRL优先释放是泌乳细胞群体内功能异质性的结果。合成速率和对TRH反应性的主要差异说明了功能异质性。
