EXOCYTOSIS IN NORMAL ANTERIOR-PITUITARY CELLS - QUANTITATIVE CORRELATION BETWEEN GROWTH-HORMONE RELEASE AND THE MORPHOLOGICAL FEATURES OF EXOCYTOSIS
EXOCYTOSIS IN NORMAL ANTERIOR-PITUITARY CELLS - QUANTITATIVE CORRELATION BETWEEN GROWTH-HORMONE RELEASE AND THE MORPHOLOGICAL FEATURES OF EXOCYTOSIS
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DOI：
10.1172/jci113415
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发表时间：
1988-04-01
影响因子：
15.9
通讯作者：
STAEHELIN, LA
中科院分区：
文献类型：
--
作者：
DRAZNIN, B;DAHL, R;STAEHELIN, LA
我们采用高压冷冻技术研究了大鼠垂体前叶细胞的胞吐现象。细胞不受刺激或暴露于1 nM生长激素释放因子（GRF）中10 min，然后进行超快速冷冻。生长激素（GH）释放的大小与冷冻断裂电镜观察到的胞外事件的数量相关。高压冷冻未固定和未冷冻保护的标本允许冷冻样品高达1毫米直径（0.5毫米厚）没有冰晶损坏，并在10毫秒内阻止胞外事件。我们的研究比较了常规固定的标本和高压冷冻的标本，证实了在潜在的胞外吞噬部位的膜内颗粒清除区域是常规固定和/或冷冻保护技术的产物。暴露于1 nM GRF下的细胞比未受刺激的细胞多释放约5倍的GH。形态学上，我们观察到grf刺激细胞的胞外事件数量增加了3.3倍，达到33.7个事件/100亩。M2与10.4件/100亩相比。M2为未受刺激的细胞。在其他实验中，我们研究了两种抑制grf诱导的胞吐作用，生长抑素和异硫酸钠。两种化合物引起相同的反应，胞外事件和分泌产物的平行减少。我们得出结论，高压冷冻，结合冷冻断裂和冷冻替代处理技术，是研究胞外分泌形态学方面的一个很好的工具。在目前的研究中，它使我们能够定量地联系垂体前叶细胞胞吐的生化和形态学。
We have used high-pressure freezing techniques to study exocytosis in rat anterior pituitary cells. The cells were either unstimulated or exposed to 1 nM growth hormone releasing factor (GRF) for 10 min before ultrarapid freezing. The magnitude of growth hormone (GH) release was then correlated with the number of exocytotic events observed with freeze-fracture electron microscopy. High-pressure freezing of unfixed and uncryoprotected specimens permits cryofixation of samples up to 1 mm diam (0.5 mm thick) without ice crystal damage, and arrests exocytotic events within 10 ms. Our studies comparing conventionally fixed specimens with those prepared by high-pressure freezing confirm that areas of intramembrane particle clearing at potential exocytotic sites are an artifact of conventional fixation and/or cryoprotective techniques. The cells exposed to 1 nM GRF released approximately fivefold more GH than did unstimulated cells. Morphologically, we have observed a 3.3-fold increase in the number of exocytotic events in GRF-stimulated cells, 33.7 events/100.mu.m2 compared with 10.4 events/100 .mu.m2 for unstimulated cells. In additional experiments, we studied the effects of two inhibitors of GRF-induced exocytosis, somatostatin and sodium isethionate. Both compounds elicit the same response, a parallel decrease in exocytotic events and in secreted product. We conclude that high-pressure freezing, combined with freeze-fracture and freeze-substitution processing techniques, is an excellent tool for studying the morphological aspects of exocytosis. In the present investigation, it has allowed us to quantitatively relate the biochemistry and morphology of exocytosis in anterior pituitary cells.