- 學位論文
花生四烯酸致使幼鼠心肌細胞凋亡之機制探討
The Mechanism of Arachidonic Acid Induced Apoptosis in Rat Cardiomyocytes.
摘要
Arachidonic acid（AA；花生四烯酸）對於體內各細胞之生理弁鄏閉蛪篲s泛的影響。除可做為eicosanoids（類二十碳酸）之前趨物質外，AA亦可直接或者間接地藉由其下游代謝產物來影響細胞膜上的離子通道、活化PKC（protein kinase C）、調節神經傳導物質的釋放和回收、促進神經元本身及其樹突的生長。平時，細胞內具有良好的調控機制可將AA維持在適當的濃度。然而，在細胞遭受缺血性傷害（ischemia）時，會活化PLC （phospholipase C）及PLA2（phospholipase A2），再加上此時ATP的生成減少使得酯化作用受阻，於是AA聚集而促使大量自由基產生，進而對細胞造成傷害。細胞內鈣離子的
並列摘要
Arachidonic acid (AA) and its metabolites have been shown to have important physiological or patho-physiological functions in many cells. Cellular calcium overload is an important determinant of ischemic myocardial injury. Over recent years, mitochondrial calcium overload has also been thought to be critical in the pathogenesis of irreversible ischemic cell death. The existence of a pathway that allows mitochondria to accumulate calcium has been established for many years. Recent studies showed that AA induced calcium influx in many different cells, but the mechanism is unclear. In the present study, we have used microfluorometry to explore the interplay between AA and cytosolic calcium (monitored using Fura-2) 、cytosolic sodium (monitored using SBFI) in primary culture of rat cardiomyocytes. Addition of AA induced a slow but sustained rise in cytosolic calcium concentration（[Ca2+]c）and cytosolic sodium concentration（[Na+]c）. The magnitude of the rise in [Ca2+]c and [Na+]c increase with the concentration of applied AA. Which means the rise in the [Ca2+]c and [Na+]c induced by AA is dose-dependent. Using patch clamp technique , we showed that AA-induced Ca2+ entry is mediated via a non-selective cation channel. To get better resolution of the subcellular functional organization of calcium signaling, we used the time-lapse confocal recording of live cardiomyocytes to observe the changes of mitochondrial calcium concentration ([Ca2+] m, monitored by Rhod-2) and mitochondrial sodium concentration ([Na+] m, monitored by CoroNa Red) induced by AA. Results showed that AA induced a substantial increase of [Ca2+] c and [Na+] c, followed immediately by a rise in [Ca2+]m and [Na+]m. We monitored the mitochondrial membrane potential (△Ψm) by TMRM and the morphology of mitochondrial by MTG. AA treatment collapsed the △Ψm and induced the structural change of mitochondria. It has been known that the △Ψm depolarization result in opening of the permeation transition pore (PTP), followed by cytochrome c release, which is the upper signal of the programmed cell death pathway. ROS augmentation and lysosome disruption can also be detected after AA treatment. We have examined the AA-induced apoptotic machinery (including cytochrome c release, caspase activation and apoptosis) in primary culture of rat cardiomyocytes with immunofluorescence technique, and we have found that the AA-induced apoptotic machinery partially was inhibited by Ca-free、Na-free、antioxidant and pepstatin A. Our findings suggest that AA activates a non-selective cation channel, resulting in calcium influx, mitochondrial calcium overload and deporlarization, cytochrome c release and apoptosis.