title: Flow cytometric analysis of ca-induced membrane permeability transition of isolated rat liver mitochondria.
abstract: The membrane permeability transition (MPT) of mitochondria plays an important role in the mechanism of apoptotic cell death in various cells. Classic type MPT is induced by Ca(2+) in the presence of inorganic phosphate and respiratory substrate, and is characterized by various events including generation of reactive oxygen species (ROS), membrane depolarization, swelling, release of Ca(2+) and high sensitivity to cyclosporine A. However, the sequence of these events and the effect of antioxidants on their events remain obscure. Flow cytometry is a convenient method to investigate the order of events among various functions occurring in MPT using a limited amount of mitochondria (200 microl of 0.02 mg protein/ml) without contamination by other organelles. Flow cytometric analysis revealed that Ca(2+) sequentially induced ROS generation, depolarization, swelling and Ca(2+) release in mitochondria by a cyclosporine A-inhibitable mechanism. These results were supported by the finding that Ca(2+)-induced MPT was inhibited by antioxidants, such as glutathione and N-acetylcysteine. It was also revealed that various inhibitors of Ca(2+)-induced phospholipase A(2) suppressed all of the events associated with Ca(2+)-induced MPT. These results suggested that ROS generation and phospholipase A(2) activation by Ca(2+) underlie the mechanism of the initiation of MPT.
authors.affiliation: Department of Cytology & Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikatacho, Okayama 700-8558, Japan.
authors.forename: Teruo
authors.initials: T
authors.lastname: Umegaki
authors.forename: Yuya
authors.initials: Y
authors.lastname: Okimura
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Fujita
authors.forename: Hiromi
authors.initials: H
authors.lastname: Yano
authors.forename: Jitsuo
authors.initials: J
authors.lastname: Akiyama
authors.forename: Masayasu
authors.initials: M
authors.lastname: Inoue
authors.forename: Kozo
authors.initials: K
authors.lastname: Utsumi
authors.forename: Junzo
authors.initials: J
authors.lastname: Sasaki