Flow cytometric analysis of Ca2+-induced membrane permeability transition of isolated rat liver mitochondria

Teruo Umegaki, Yuya Okimura, Hirofumi Fujita, Hiromi Yano, Jitsuo Akiyama, Masayasu Inoue, Kozo Utsumi, Junzo Sasaki

Research output: Contribution to journalArticle

5 Citations (Scopus)

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 Ca2+ 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 Ca2+ 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 μl of 0.02 mg protein/ml) without contamination by other organelles. Flow cytometric analysis revealed that Ca2+ sequentially induced ROS generation, depolarization, swelling and Ca2+ release in mitochondria by a cyclosporine A-inhibitable mechanism. These results were supported by the finding that Ca2+-induced MPT was inhibited by antioxidants, such as glutathione and N-acetylcysteine. It was also revealed that various inhibitors of Ca2+-induced phospholipase A2 suppressed all of the events associated with Ca2+-induced MPT. These results suggested that ROS generation and phospholipase A2 activation by Ca2+ underlie the mechanism of the initiation of MPT.

Original languageEnglish
Pages (from-to)35-44
Number of pages10
Journaljournal of clinical biochemistry and nutrition
Volume42
Issue number1
DOIs
Publication statusPublished - Jan 2008

Keywords

  • Antioxidant
  • Flow cytometric analysis
  • Membrane permeability transition
  • Mitochondria
  • Phospholipase A

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Nutrition and Dietetics
  • Clinical Biochemistry

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