Sirianni M J, Fujimoto K I, Nelson C S, Pellegrino M J, Allen R G
Center for Research on Occupational and Environmental Toxicology, The Oregon Health Sciences University, Portland 97201, USA.
DNA Cell Biol. 1999 Jan;18(1):51-8. doi: 10.1089/104454999315619.
Recent studies have shown that cAMP analogs can induce expression of prepro (pp) orphanin FA (OFQ)/nociceptin-related gene products in NS20Y mouse neuroblastoma cells (Saito et al. [1996]. J Biol Chem 271, 15615-15622). Additionally, exposure of NS20Y cells to cAMP analogs promoted neurite outgrowth and large dense-core vesicle formation. Even though an OFQ-like precursor (called 27K) was identified in NS20Y cell extracts, no secretion of OFQ-related peptides was detected. We have used reversed-phase high-performance liquid chromatography combined with a specific radioimmunoassay for OFQ(1-17) to determine if NS20Y cells secrete ppOFQ-derived peptides when stimulated by the cAMP analog ctp-cAMP. We found that NS20Y cells secreted abundant amounts of OFQ-derived products when stimulated by cAMP analogs. We also have determined that secretion of OFQ peptides was both time and concentration dependent and reversible on removal of cAMP analogs from the culture medium. In addition, the opioid agonist D-Pen2-D-Pen5-enkephalin inhibited forskolin-stimulated OFQ peptide secretion. Further, the synthetic glucocorticoid dexamethasone virtually abolished ctp-cAMP-stimulated OFQ peptide secretion. These results suggest that the biosynthesis, processing, and secretion of the OFQ neuropeptide transmitter system can be modulated through intracellular cAMP levels and that these functions are regulated by opioids and molecules involved in mediating the stress response. The NS20Y cell system will be extremely valuable for studying the regulation of OFQ-derived peptides by a variety of intra-cellular and extracellular signaling pathways.
最近的研究表明，环磷酸腺苷（cAMP）类似物可诱导NS20Y小鼠神经母细胞瘤细胞中前阿片样物质原（pp）孤啡肽（OFQ）/孤啡肽原（nociceptin）相关基因产物的表达（斋藤等人，[1996年]。《生物化学杂志》271，15615 - 15622）。此外，将NS20Y细胞暴露于cAMP类似物可促进神经突生长和大的致密核心囊泡形成。尽管在NS20Y细胞提取物中鉴定出一种类似OFQ的前体（称为27K），但未检测到OFQ相关肽的分泌。我们使用反相高效液相色谱结合针对OFQ(1 - 17)的特异性放射免疫分析法，来确定当受到cAMP类似物ctp - cAMP刺激时，NS20Y细胞是否分泌源自ppOFQ的肽。我们发现，当受到cAMP类似物刺激时，NS20Y细胞分泌大量源自OFQ的产物。我们还确定，OFQ肽的分泌既具有时间依赖性又具有浓度依赖性，并且在从培养基中去除cAMP类似物后是可逆的。此外，阿片样物质激动剂D - Pen2 - D - Pen5 - 脑啡肽抑制了福斯可林刺激的OFQ肽分泌。此外，合成糖皮质激素地塞米松实际上消除了ctp - cAMP刺激的OFQ肽分泌。这些结果表明，OFQ神经肽递质系统的生物合成、加工和分泌可通过细胞内cAMP水平进行调节，并且这些功能受阿片样物质以及参与介导应激反应的分子调控。NS20Y细胞系统对于研究多种细胞内和细胞外信号通路对源自OFQ的肽的调节将具有极高的价值。
