The Mechanism of Arachidonic Acid Induced Apoptosis in Rat Cerebellar Granule Cells

• Main Authors: Chia-Hao Hsu, 許家豪
• Other Authors: 吳美玲
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/03640704127316251810

碩士 === 國立臺灣大學 === 生理學研究所 === 92 === Abstract The ischemic injury is associated with severe cellular injury in stroke, seizure, Alzheimer’s disease and many other degenerative disorders of nervous system. Reperfusion after brain ischemia results in disorder of energy supply, the perturbation of calcium homeostasis, the production of arachidonic acid; oxyradicals and nitric oxide, and activation of many kinases and prostaglandins of nervous system, leading to the death of nervous cell. Arachidonic acid (AA) and its metabolites have been shown to have important physiological or patho-physiological functions in nervous system. The death of neurons can be divided into two categories: apoptosis and necrosis. In the present study, we especially focus on the apoptosis. Of many pathways inducing apoptosis, one of them is related to mitochondria. This pathway initiates at the release of cytochrome c from the mitochondrial permeation transition pore, then cytochrome c triggers the activation of caspases cascade, resulting in the condensation of chromatin and fragmentation of DNA. However, the relationship between AA abundantly produced and apoptosis during ischemia injury is still unclear. In the present study, we applied AA (10 �嵱) to mimic the conditions of ischemic reperfusion injury. Using immunocytochemistry staining techniques, we found that the release of cytochrome c, activation of caspase, and apoptosis in the cerebellar granule cell all increased after AA treatment. However, after pretreatment of calicium-free solution, these effects were all inhibited. Therefore it was suggested that apoptosis is closely related to the cellular calcium concentration ([Ca2+] c). We used microspectrofluorometry (monitored by fura-2) to examine the change of [Ca2+] c in the cerebellar granule cells after the AA treatment. We showed that AA induced a substantial increase of [Ca2+] c in the cerebellar granule cells, and the response is proportional to the concentration of AA. We used confocal microscopy techniques (monitored by rhod-2) to detect mitochondrial calcium ([Ca2+] m). We found that a rise in [Ca2+] c was followed by a rise in [Ca2+] m. Using Ru360 to inhibit the mitochondrial calcium uptake through calcium uniporter, we observed that a rise in [Ca2+] m was inhibited. The mitochondrial membrane potential (△Ψm) was monitored with TMRM. After the addition of AA, △Ψm decreased immediately. Thus, we conclude that, AA treatment in cerebellar granule cells, results in calcium influxs, calcium entry into mitochondria through calcium uniporter, leading to a rise in [Ca2+] m, besides we observe the depolarization of mitochondria, opening of mitochondrial permeation transition pore, causing the release of cytochrome c, activation of caspases, and apoptosis.