Oxidative damage to mitochondria is mediated by the Ca2+-dependent inner membrane permeability transition

Research Project

Project/Area Number	06454376
Research Category	Grant-in-Aid for General Scientific Research (B)
Allocation Type	Single-year Grants
Research Field	General surgery
Research Institution	Kansai Medical University
Principal Investigator	TANAKA Takaya Kansai Medical University, 医学部, 助教授 (70121952)
Co-Investigator(Kenkyū-buntansha)	原田直己関西医科大学, 医学部, 助手 (10121972)
Project Period (FY)	1994 – 1995
Project Status	Completed (Fiscal Year 1995)
Budget Amount *help	¥3,300,000 (Direct Cost: ¥3,300,000) Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000) Fiscal Year 1994: ¥2,300,000 (Direct Cost: ¥2,300,000)
Keywords	Anoxia / recoxygenation / cyclosporin A / DiOC6 / JC-1 / thapsigargin / mitochondrial membrane potential / permeability transition / JC-l / ruthnium red
Research Abstract	The aim of this study is to evaluate changes in mitochondrial function of cultured MDCK cell by laser scanning confocal microscopy and a digitized-fluorescence-imaging during chemical anoxia/reoxygenation (A/R). Chemical anoxia was induced with rotenone and 2-deoxyglucose, inhibitor of a site I of the electron transport chain and glycolysis, respectively. Oxidative metabolism was restored by the addition of a short odd-chain fatty acid (heptanoate). Cytosolic free Ca^<2+> measured by Futa-2 increased gradually about 50% after 30 min of anoxia and returned to initial value immediately after reoxygenation. Anoxia up to 60 min or 30 min of anoxia followed by 30 min of reoxygenation had no effect on cell viability and mitochondrial membrane potential (DELTAPSI_m) assessed by DiOC6 and JC-1. In the presence of thapsigargin, a specific inhibitor of the Ca-ATPase of the endoplasmic reticulum, DELTAPSI_m was stable during 60 min of anoxia. However, DELTAPSI_m decreased with reoxygenation after 30 min of anoxia. The sensitization of A/R by thapsigargin disappeared in the presence of cyclosporin A,a specific inhibitor of mitochondrial inner membrane pore opening. We conclude that reoxygenation-induced Ca^<2+> redistribution and the mitochondrial membrane permeability transtion are involved in A/R injury of MDCK cell.