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Cyclic AMP analogs induce synthesis, processing, and secretion of prepro nociceptin/orphanin FQ-derived peptides by
M J Sirianni1, K I Fujimoto, C S Nelson
1Center for Research on Occupational and Environmental Toxicology, The Oregon Health Sciences University, Portland 97201, USA.
Abstract:
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.
Insights
Cyclic AMP (cAMP) analogs stimulate the secretion of orphanin (OFQ)-derived peptides from neuroblastoma cells. This secretion is regulated by opioids and stress response molecules, offering a valuable model for studying neuropeptide signaling.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Recent studies indicate cAMP analogs induce expression of prepro (pp) orphanin FA (OFQ)/nociceptin-related gene products in NS20Y mouse neuroblastoma cells.
- Exposure to cAMP analogs promotes neurite outgrowth and dense-core vesicle formation in NS20Y cells.
- Previous research identified an OFQ-like precursor (27K) in NS20Y cells, but no OFQ-related peptide secretion was detected.
Purpose of the Study:
- To investigate whether NS20Y cells secrete ppOFQ-derived peptides when stimulated by the cAMP analog ctp-cAMP.
- To characterize the regulation of OFQ peptide secretion by intracellular cAMP levels, opioids, and stress response mediators.
Main Methods:
- Utilized reversed-phase high-performance liquid chromatography (RP-HPLC).
- Employed a specific radioimmunoassay for OFQ(1-17).
- Stimulated NS20Y cells with cAMP analogs and measured peptide secretion.
Main Results:
- NS20Y cells secreted abundant OFQ-derived products upon stimulation with cAMP analogs.
- Secretion was time- and concentration-dependent and reversible upon removal of cAMP analogs.
- Opioid agonist D-Pen2-D-Pen5-enkephalin inhibited secretion, while dexamethasone abolished it.
Conclusions:
- Intracellular cAMP levels modulate the biosynthesis, processing, and secretion of the OFQ neuropeptide transmitter system.
- OFQ peptide secretion is regulated by opioids and molecules involved in the stress response.
- The NS20Y cell system is a valuable model for studying OFQ-derived peptide regulation via signaling pathways.