The Expression and Mechanism of Inducible Nitric Oxide Synthase in Hippocampus of Rats under Normal Light-Dark Cycle

• Main Authors: Chia-Yu Hsieh, 謝家瑜
• Other Authors: Jia-Yi Wang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/16512892870989349014

碩士 === 臺北醫學大學 === 醫學科學研究所 === 98 === Abstract Suprachiasmatic nucleus (SCN) is the circadian pacemaker of the brain. This clock can be modulated by afferent inputs in response to a variety of external and internal cues. Recent studies showed that cAMP response element binding protein (CREB) and neuronal nitric oxide synthase (nNOS) are rhythmically expressed in SCN. On the other hand, circadian rhythm disturbance induced expression of inducible NOS (iNOS). However, how iNOS expressed rhythmicallly in normal circadian rhythm remains unknown. We explored the circadian rhythm of iNOS expression by RT-PCR in detection of iNOS mRNA in cerebral cortex, hippocampus and peripheral blood；Western blot analysis was used for determination of iNOS protein and phosphorylated CREB under a 12-hour light / dark cycle and 24-hour darkness condition. The chromatin immunoprecipitation (ChIP) assay was used for figuring out the transcriptional event of iNOS expression. The results showed that the rhythms of iNOS mRNA expression had increasing peaks at ZT 6 and ZT 22, and iNOS protein level also had rhythmically expression with peak at ZT 18. Remarkably, iNOS protein levels were increased in light phase after ZT 22 which is the time of the increasing peak of iNOS mRNA. Furthermore, phospho-CREB, the activated form of CREB, had higher expressed levels at ZT 18 and ZT 22, and in ChIP assay showed that phospho-CREB bound to iNOS promoter at ZT 18. Whereas, there had no significant changes on phospho-CREB and iNOS under the 24-hour darkness condition without light-darkness shifting. Interestingly, we found that the level of iNOS mRNA in hippocampus is closely parallel and correlated with the level of iNOS mRNA in the peripheral blood. In conclusion, we demonstrated that iNOS is rhythmically expressed in hippocampus during normal light-dark cycle and it seems to be mediated by the activation of CREB, which might be affected by light-dark cycle.