Investigation of oATP-induced changes in mitochondria-mediated cell death

• Main Authors: Hsiang-Wei Hsing, 邢翔威
• Other Authors: Synthia H. Sun
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/59735365613903461000

碩士 === 國立陽明大學 === 神經科學研究所 === 102 === Our early results have shown that prolonged treatment of N2a neuroblastoma cells with a subthreshold concentration (50 M) of P2X7R antagonist, periodate-oxidized ATP (oATP), decreased cell viabilities and induced apoptosis. This study is aiming to elucidate the possible mechanisms involved. Our results showed that prolonged treatment of 25-100 M oATP decreased cell viabilities. In addition, the oATP-decreased cell viabilities were exacerbated in the P2X7R knockdown N2a cells. We then over expressed a C-terminal trunketed P2X7R in these cells. Our results further confirmed that oATP might mediate both P2X7R-dependent and P2X7R-independent pathway to decrease cell viabilities. To investigate the involvement of Ca2+ signaling, we increased intracellular Ca2+ concentration ([Ca2+]i) with ionomycin. Our results revealed that 0.025-0.2 M ionomicin induced transient increases in [Ca2+]I but have no effect on oATP-decreased cell viability. Nevertheless, 3 M ionomicin induced a sustained increase in [Ca2+]I and exacerbated the oATP-decreased cell viability. Thus, the decreases in cell viability caused by ionomycin and oATP might mediate through distinct mechanisms. Therefore, we examine the effect of oATP on mitochondria morphology. Our results revealed that treatment of N2a cells with oATP and a known apoptosis inducer, staurosporine, induced mitochondria clustering as labeled by MitoTracker Red. In addition, oATP induced cytochrome C release. We also observed that oATP and staurosporine both increased the cell number of a mitochondrial specific Ca2+ dye, X-Rhod1, stained cells. Thus oATP might increase Ca2+ concentration and damage mitochondria. Taken together, our results demonstrated that oATP might affect mitochondria activities of N2a neuroblastoma cells and that was P2X7R-independent.