Related Experiment Video
Updated: Jul 7, 2026
Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria
Published on: September 7, 2012
Flow cytometric analysis of ca-induced membrane permeability transition of isolated rat liver mitochondria
Teruo Umegaki1, Yuya Okimura, Hirofumi Fujita
1Department of Cytology & Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikatacho, Okayama 700-8558, Japan.
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.
Insights
Calcium (Ca2+) induces mitochondrial permeability transition (MPT) by first generating reactive oxygen species (ROS), then causing depolarization, swelling, and Ca2+ release. Antioxidants and phospholipase A2 inhibitors block these MPT events.
Area of Science:
- Mitochondrial biology
- Cell death mechanisms
- Biochemistry
Background:
- Mitochondrial permeability transition (MPT) is crucial in apoptotic cell death.
- Classic MPT involves Ca2+, phosphate, and substrates, leading to ROS, depolarization, swelling, and Ca2+ release.
- The precise sequence of MPT events and antioxidant effects are not fully understood.
Purpose of the Study:
- To elucidate the sequential events of calcium-induced MPT in mitochondria.
- To investigate the role of reactive oxygen species (ROS) and antioxidants in MPT.
- To explore the involvement of phospholipase A2 in MPT.
Main Methods:
- Utilized flow cytometry for precise analysis of MPT events in isolated mitochondria.
- Employed antioxidants like glutathione and N-acetylcysteine to assess their inhibitory effects.
- Tested various inhibitors of calcium-induced phospholipase A2.
Main Results:
- Flow cytometry demonstrated a sequential induction of ROS generation, depolarization, swelling, and Ca2+ release by Ca2+.
- These Ca2+-induced events were sensitive to cyclosporine A and inhibited by antioxidants.
- Inhibitors of Ca2+-induced phospholipase A2 effectively suppressed all observed MPT events.
Conclusions:
- Calcium-induced MPT proceeds sequentially through ROS generation, depolarization, swelling, and Ca2+ release.
- ROS generation and phospholipase A2 activation are key mechanisms initiating Ca2+-induced MPT.
- Antioxidants and phospholipase A2 inhibitors offer potential therapeutic targets for modulating MPT.
