Mechanism of induction of hypertonicity-induced apoptosis

2003 Fiscal Year Final Research Report Summary

• Project/Area Number: 14571022
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Kidney internal medicine
• Research Institution: Osaka University
• Principal Investigator: HORIO Masaru HORIO,Masaru, 医学系研究科, 助教授 (20273633)
• Project Period (FY): 2002 – 2003
• Keywords: hypertonicity / mitochondria / peritoneal mesothellial cells / cytochrome c / apoptosis / cyclosporin A / caspase

Research Abstract

Mechanism of hypertonicity-induced apoptosis in cultured peritoneal mesothelial cells was investigated. When the cell were exposed to 700 mOsm medium made by addition of glucose, apoptosis was demonstrated within 8 h. Cytochrome c was released from mitochondria within 2h, followed by activation of caspase-3 activity, suggesting that mitochondrial pathway plays an important role in hypertonicity-induced apoptosis. Protein level of p53 was decreased rapidly and protein levels of Bax, Bcl-2, and Bcl-XL were unchanged. Therefore involvement of these proteins in th release of cytochrome c seems to be little. Using isolated mitochondria, direct effects of hypertonicity on the size of mitochondoria and release of cytochrome c were studied. Hypertonic medium made by addition of KC1 or glucose reduced the size of mitochondria similarly. On the other hand, cytochrome c release from isolated mitochondria was demonstrated only when KC1 or NaC1 was used as an osmotic agent. Cytochrome c release was not demonstrated by hypertonic medium made by addition of glucose, sucrose, or mannitol. These results indicated that the release of cytochrom c was not due to hypertonic mechanical damage of the mitochondrial membrane, but might be due to high ionic strength. Simultaneous addition of 0.02mM cyclosporin A (CsA) inhibited the KC1-induced release of cytochrome c from isolated mitochondria. When the cells were exposed to 700 mOsm medium, CsA inhibited the hypertonicity-induced activation of caspase-3 in a dose dependent manner. These results suggest the involvement of CsA-sensitive mitochondrial permeability transition pore in the hypertonicity-induced cytochrome c release. It has been known that intracellular concentrations of electrolytes increase rapidly after hypertonic exposure, then the electrolytes are replaced by compatible organic osmolytes. Initial high concentrations of intracellular electrolytes could damage mitochondria, leading to the cytochrome c release, a trigger of caspase activation cascade.