MacArthur L, Iacangelo A L, Hsu C M, Eiden L E
Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20892.
J Physiol Paris. 1992;86(1-3):89-98. doi: 10.1016/s0928-4257(05)80012-9.
The molecular mechanisms regulating neuropeptide and secretory protein biosynthesis in neuroendocrine cells were examined using the prototype neuropeptide and secretory proteins enkephalin and chromogranin A (CGA). Treatment with the secretogogue nicotine results in the calcium-dependent secretion of enkephalin peptides from bovine chromaffin cells in primary culture and a concomitant increase in enkephalin peptide biosynthesis. Both secretion and biosynthesis are also stimulated by cell depolarization with elevated potassium. Elevation of intracellular cyclic AMP, on the other hand, results in stimulation of enkephalin biosynthesis and long-term, but not acute, secretion of enkephalin peptides. Coupling of enkephalin biosynthesis to calcium influx occurs at the level of transcription of the enkephalin gene. Thus, potassium depolarization causes a calcium-dependent elevation of enkephalin mRNA which is preceded by an increase in the rate of transcription of the enkephalin gene in the chromaffin cell. The accumulation of enkephalin message or peptide by potassium depolarization or treatment with nicotine is prevented by D600 or hexamethonium respectively, added 1 h after addition of nicotine or KCl and following acute release, suggesting that calcium acts as a continuous rather than triggering stimulus for enkephalin biosynthesis. Sequence analysis of the bovine enkephalin promoter identified sequence conservation of three enhancers previously reported in the human gene which are required for regulation of the gene by calcium, cAMP, and phorbol ester in vitro. In contrast to the regulation of the enkephalin system, no increase in either CGA or CGB mRNA or gene transcription attended depolarization-induced secretion from chromaffin cells. Since enkephalin and CGA are co-stored in and co-released from the same secretory vesicles in these cells, the results imply that a surplus of CGA is constitutively synthesized in chromaffin cells such that compensatory up-regulation during changes in the secretory state of the cell, such as occurs for enkephalin, is not required for the secretory proteins.
利用神经肽和分泌蛋白脑啡肽及嗜铬粒蛋白A（CGA）这两种典型物质，研究了神经内分泌细胞中调节神经肽和分泌蛋白生物合成的分子机制。促分泌剂尼古丁处理可导致原代培养的牛嗜铬细胞中脑啡肽肽段的钙依赖性分泌，并伴随脑啡肽肽段生物合成的增加。细胞用高钾进行去极化也会刺激分泌和生物合成。另一方面，细胞内环状AMP水平的升高会刺激脑啡肽的生物合成以及脑啡肽肽段的长期（而非急性）分泌。脑啡肽生物合成与钙内流的偶联发生在脑啡肽基因转录水平。因此，钾去极化会导致脑啡肽mRNA的钙依赖性升高，在此之前嗜铬细胞中脑啡肽基因的转录速率会增加。在添加尼古丁或氯化钾1小时后并在急性释放后分别添加D600或六甲铵，可分别阻止钾去极化或尼古丁处理导致的脑啡肽信息或肽段的积累，这表明钙作为脑啡肽生物合成的持续刺激而非触发刺激。牛脑啡肽启动子的序列分析确定了先前在人类基因中报道的三个增强子的序列保守性，这些增强子在体外对于该基因受钙、cAMP和佛波酯的调节是必需的。与脑啡肽系统的调节不同，嗜铬细胞去极化诱导的分泌并未伴随CGA或CGB mRNA或基因转录的增加。由于在这些细胞中脑啡肽和CGA共同储存于同一分泌囊泡并共同释放，结果表明嗜铬细胞中组成性合成了过量的CGA，使得在细胞分泌状态发生变化时（如脑啡肽那样）不需要像分泌蛋白那样进行代偿性上调。
