Childs G V, Lloyd J M, Rougeau D, Unabia G
Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston 77550.
Endocrinology. 1988 Dec;123(6):2885-95. doi: 10.1210/endo-123-6-2885.
Anterior pituitary corticotropes represent only 9-10% of the mixed pituitary cell population. However, their small size precludes their enrichment because they cannot be separated from the more abundant PRL and GH cells. They can be induced to enlarge by adrenalectomy, and this report describes the separation of larger CRH-responsive corticotropes from a subpopulation of small pituitary cells. The separation was done by counterflow centrifugation in an elutriator containing the Sanderson chamber which was designed to separate small cells under 15 micron in diameter. The corticotropes were initially eluted at flow rates under 30 ml/min along with other cells less than 12.5 micron in diameter. They were then stimulated for 2-4 h with 0.5 nM CRH and reeluted with the use of higher flow rates to separate the enlarged corticotropes from the unstimulated cells. Reelutriation of the entire pool of small cells produced an enrichment to 60% corticotropes in five separate experiments. However, when the pool was divided into smaller cells (eluted at 20 ml/min) and medium-sized cells (eluted at 30 ml/min), and the two pools were reeluted separately, the enrichment increased to over 90% corticotropes in eight separate experiments. These corticotrope populations remained enriched for up to 14 days in culture. They also secreted in a reverse hemolytic plaque assay that recognizes ACTH-(25-39). The dual labels for ACTH and beta-endorphin showed that 60% of the corticotropes stored both peptides, whereas 30% stored only ACTH, and 10% stored only beta-endorphin. No differences in storage patterns were seen when small and medium-sized corticotropes were compared. Thus, these studies present the first report of the production of an enriched fraction of CRH-responsive corticotropes by counterflow centrifugation and the first report of heterogeneous storage of ACTH and beta-endorphin. The use of enriched fractions facilitated the analysis of these heterogeneous storage patterns in over 8000 corticotropes.
垂体前叶促肾上腺皮质激素细胞仅占垂体混合细胞群的9 - 10%。然而，由于它们体积小，无法从数量更多的催乳素细胞和生长激素细胞中分离出来进行富集。肾上腺切除可诱导它们增大，本报告描述了从一小部分垂体小细胞亚群中分离出对促肾上腺皮质激素释放激素（CRH）反应性更强的促肾上腺皮质激素细胞的方法。分离是在一个装有桑德森室的淘洗器中通过逆流离心进行的，该淘洗器旨在分离直径小于15微米的小细胞。促肾上腺皮质激素细胞最初与其他直径小于12.5微米的细胞一起以流速低于30毫升/分钟被洗脱。然后用0.5纳摩尔/升的CRH刺激它们2 - 4小时，再使用更高的流速进行再洗脱，以从未受刺激的细胞中分离出增大的促肾上腺皮质激素细胞。在五个独立实验中，对整个小细胞池进行再淘洗后，促肾上腺皮质激素细胞富集到了60%。然而，当将细胞池分为较小细胞（以20毫升/分钟洗脱）和中等大小细胞（以30毫升/分钟洗脱），并分别对这两个细胞池进行再淘洗时，在八个独立实验中，促肾上腺皮质激素细胞的富集率增加到了90%以上。这些促肾上腺皮质激素细胞群体在培养中可保持富集状态长达14天。它们在一种识别促肾上腺皮质激素（ACTH）-（25 - 39）的反向溶血空斑试验中也有分泌。ACTH和β - 内啡肽的双重标记显示，60%的促肾上腺皮质激素细胞储存了这两种肽，而30%仅储存ACTH，10%仅储存β - 内啡肽。比较小和中等大小的促肾上腺皮质激素细胞时，未发现储存模式有差异。因此，这些研究首次报道了通过逆流离心产生富集的对CRH反应性促肾上腺皮质激素细胞组分，以及首次报道了ACTH和β - 内啡肽的异质性储存。使用富集组分便于对8000多个促肾上腺皮质激素细胞中的这些异质性储存模式进行分析。
