| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Hypoxia/ischemia is a potent stimulator of myocyte apoptosis. The precise mechanism by which hypoxia induces apoptosis remains still unclear, however, recent reports have proposed the possibility that cellular acidification plays a crucial role in this process. We have already demonstrated that hypoxia induces myocytes apoptosis via mitochondrial death pathway in the presence of ATP and their triggers are Bax translocation. The present study was, therefore, designed to examine whether acidification induces apoptosis via the mitochondrial pathway and the roles of Bax and mitochondrial permeability transition during acidosis in cardiac myocytes. Neonatal rat cardiac myocytes were incubated in modified Tyrode solution for 7h in which the pH was adjusted to 7.4,7.2,6.8,or 6.5. Extracellular acidification increased the percentage of apoptotic myocytes in a pH-dependent manner, such that pH 7.4,7.2,6.8,and 6.5 resulted in 3.8±0.5,4.7±0.4,6.9±0.3,9.2±6.2% of apoptotic cells, respectively. Mor
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eover, intracellular acidification, translocation of Bax, a pro-apoptotic protein, from cytosol to mitochondria, opening of mitochondrial permeability transition pore, loss of mitochondrial membrane potential, cytochrome c release from mitochondria into cytosol, and activation of caspase-9 and 3 were observed in a pH-dependent fashion. In order to examine whether intracellular acidification also directly provokes Bax translocation and mitochondrial permeability transition, we exposed cell suspension to acidification, performed immunoblotting, and monitored mitochondrial permeability transition. Likewise, Bax translocation and mitochondrial permeability transition were observed in a pH-dependent manner. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated acidosis-induced apoptosis by inhibiting mitochondrial permeability transition pore opening-associated release of cytochrome c and activation of downstream caspases. The results suggest that cellular acidification can directly evoke Bax translocation and mitochondrial permeability transition, which activate myocyte apoptosis through the mitochondrial death signaling pathway. Less
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