We have developed a novel method for monitoring the mitochondrial permeability transition in single intact hepatocytes during injury with t-butylhydroperoxide (t-BuOOH). Cultured hepatocytes were loaded with the fluorescence probes, calcein and tetramethylrhodamine methyl ester (TMRM). Depending on loading conditions, calcein labelled the cytosolic space exclusively and did not enter mitochondria or it stained both cytosol and mitochondria. TMRM labelled mitochondria as an indicator of mitochondrial polarization. Fluorescence of two probes was imaged simultaneously using laser-scanning confocal microscopy. During normal incubations, TMRM labelled mitochondria indefinitely (longer than 63 min), and calcein did not redistribute between cytosol and mitochondria. These findings indicate that the mitochondrial permeability transition pore (‘megachannel’) remained closed continuously. After addition of 100 microM t-BuOOH, mitochondria filled quickly with calcein, indicating the onset of mitochondrial permeability transition. This event was accompanied by mitochondrial depolarization, as shown by loss of TMRM. Subsequently, the concentration of ATP declined and cells lost viability. Trifluoperazine, a phospholipase inhibitor that inhibits the permeability transition in isolated mitochondria, prevented calcein redistribution into mitochondria, mitochondrial depolarization, ATP depletion and cell death. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, also rapidly depolarized mitochondria of intact hepatocytes but did not alone induce a permeability transition. Trifluoperazine did not prevent ATP depletion and cell death after the addition of CCCP. In conclusion, the permeability transition pore does not ‘flicker’ open during normal incubation of hepatocytes but remains continuously closed. Moreover, mitochondrial depolarization per se does not cause the permeability transition in intact cells. During oxidative stress, however, a permeability transition occurs quickly which leads to mitochondrial depolarization and cell death.
Skip Nav Destination
Article navigation
April 1995
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkAdvertising
Research Article|
April 01 1995
Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide
A L Nieminen;
A L Nieminen
*Department of Anatomy, Case Western Reserve University, Cleveland, OH 44106-4938, U.S.A.
Search for other works by this author on:
A K Saylor;
A K Saylor
†Laboratories for Cell Biology, Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, U.S.A.
Search for other works by this author on:
S A Tesfai;
S A Tesfai
†Laboratories for Cell Biology, Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, U.S.A.
Search for other works by this author on:
B Herman;
B Herman
†Laboratories for Cell Biology, Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, U.S.A.
Search for other works by this author on:
J J Lemasters
J J Lemasters
†Laboratories for Cell Biology, Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, U.S.A.
Search for other works by this author on:
Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1995 The Biochemical Society, London
1995
Biochem J (1995) 307 (1): 99–106.
Citation
A L Nieminen, A K Saylor, S A Tesfai, B Herman, J J Lemasters; Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide. Biochem J 1 April 1995; 307 (1): 99–106. doi: https://doi.org/10.1042/bj3070099
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.