Roles of Glutamate and Opioid in Hypoxia Induced Neuronal Damage

• Main Author: 林琦鑫
• Other Authors: 葛其梅
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/42658823864967609500

碩士 === 國立臺北大學 === 生物化學研究所 === 88 === The primary goal of the study was to uncover the mechanisms responsible for the hypoxia-induced neuronal death. The protective roles of glutamate and naloxone in the hypoxia-induced neuronal death were also under the investigation. The human neuroblastoma cell (SK-N-SH) and the rat primary brain cells were used in the study. Trypan able cell counting method was first used to determine the hypoxia effect on the viability of these tested cells. Mitochondria enzyme activities of the dehydrogenases and cytochrome C oxidase (COX) were further analyzed to determine the energy metabolic potential of the hypoxic cells. By analyzing the release of the lactate dehydrogenases from the hypoxic cells, we can determine whether the hypoxia-induced cell death is mediated through the necrotic pathway. On the other hand, the involvement of the appoptosis in the hypoxia was also determined by using the fluorencen-immunostaining (DAPI) method, and the electrophoresis method to analyze the features of the DNA in the hypoxic cells. To examine the effects of th eglutamates and opioid related compounds on the hypoxic cells, glutamates, opioid agonists (β-endorphins, met-enkephalins, and dynorphins), and various opioid antagonists (naloxone, nor-BIN, ICI-174, 864, and β-FNA) wer pre-delivered to the cells for 2 hrs, and then followed by 48 hrs hypoxic treatment. The effects of glutamate and naloxone on the H2O2 damaged cells were also observed, by pre-treating the cells with each drug, followed by the H2O2 treatment. 　Results showed that 48 hr hypoxia significantly damaged both the SK-N-SH and th erat primary brain cells. The mechanisms underlying the damage appeared to be mediated by interrupting the mitochondrial enzyme (dehydrogenase and COX) activities, and activating both the necrotic and apoptotic pathways in the hypoxic cells. The cytotoxic effect of glutamate (at a high concentration, such as 0.5 mM) has been reported by many group. However, we found that at a lower dose (≦ 10－6 M), glutamate showed protection on the hypoxic cells. It is believed that glutamate by acting at the non-NMDA receptor, can probable protect the hypoxic cells by elevating th mitochondrial enzyme activities (elevating the mitochondrial dehydrogenase activity in the SK-N-SH cells, and the COX activities in the rat primary brain cells), and interrupting the apoptotic activation. It is also possible that glutamate can block the cytotoxicity caused by the free radicals. The protective effect of glutamate on the hypoxic cells further suggests the absene of glutamate release during hypoxia. 　The effects of the opioid system on the hypoxic cells were also observed in the study. We found that none of the 3 major opioid types showed protection on the hypoxic cells. On contrary, the opioid antagonists, naloxone and nor-BNI showed significant protections on the hypoxic cells. The protection is through the interruption of apoptosis in the hypoxic cells. Naloxone can also block the cytotoxicity caused by the free radicals (H2O2). These results suggest that inactivation of the kappa opioid receptors is beneficial to the cells against hypoxia. The effects of naloxone or nor-BNI on the cerebral ischemia remains to be determined in the futrue study. 　In overall, results from the present study provide more information about the mechanisms involves in the hypoxia or even ischemia induced cell death. Furthermore, by using the in vitro hypoxia model, we not only can have better understanding about the mechanisms involve in the hypoxia or ischemia, but also gain a reliable way to screen for more therapeutic drugs against cerebral ischemia in the future.