Bcl-2 does not inhibit the permeability transition pore in mouse liver mitochondria

Joy C. Yang, Axel Kahn, Gino A Cortopassi

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The mechanism by which the mitochondrially-localized Bcl-2 protein inhibits apoptosis is still unclear. Some authors have proposed that apoptosis is dependent on induction of the mitochondrial permeability transition pore (PTP), and that activators of apoptosis such as Bax work through activation of PTP, whereas inhibitors of apoptosis such as Bcl-2 work through inhibition of PTP, and the consequent activation or inhibition of PTP-dependent release of mitochondrial apoptotic factors, including cytochrome c. PTP opening is classically measured by a light-scattering assay of large-amplitude swelling of rodent liver mitochondria in sucrose media. Thus to test the hypothesis that Bcl-2 inhibits either the PTP or the PTP-dependent release of cytochrome c, the rate and extent of PTP, and PTP-dependent release of cytochrome c were compared in liver mitochondria from control and Bcl-2 transgenic mice. We demonstrated that Bcl-2 protein was expressed to high levels in mitochondria of transgenics versus controls. We confirmed that while control mice undergo massive hepatic cell death upon exposure to anti-Fas antibody, the Bcl-2 transgenic livers were resistant, by the criteria of gross morphology, serum enzyme release, and caspase 3 activity. We purified mitochondria from livers of the Bcl-2 transgenics and measured PTP directly by the mitochondrial swelling assay. Purified mitochondria from both transgenics and controls were induced to undergo large-amplitude swelling that was dependent on the classical PTP inducers calcium ion (Ca2+), t-butyl hydroperoxide (tBOOH) and atractyloside (Atr); and as expected, pretreatment of mitochondria with cyclosporin A (CsA) completely abolished mitochondrial swelling. However, there was no difference in the rate or final extent of PTP induction in Bcl-2 overexpressors versus control mitochondria. Furthermore, there was no difference in the PTP dependent release of cytochrome c from Bcl-2 overexpressors versus control mitochondria. Therefore, while we observe a strong inhibition of Fas-dependent apoptosis by Bcl-2 overexpression in mouse liver, we observe no effect of Bcl-2 overexpression on either the rate or extent of mitochondrial PTP, or upon the release of cytochrome c from mitochondria in which the PTP has been induced. The simplest explanation of these results is that Bcl-2 inhibits neither PTP nor PTP-dependent release of cytochrome c, however, other possibilities are discussed. Copyright (C) 2000 Elsevier Science Ireland Ltd.

Original languageEnglish (US)
Pages (from-to)65-72
Number of pages8
JournalToxicology
Volume151
Issue number1-3
DOIs
StatePublished - Oct 26 2000

Fingerprint

Mitochondria
Liver Mitochondrion
Liver
Permeability
Cytochromes c
Swelling
Apoptosis
Assays
Mitochondrial Swelling
Atractyloside
Chemical activation
tert-Butylhydroperoxide
Cell death
Caspase 3
Light scattering
Cyclosporine
Sucrose
Proteins
Ions
Calcium

Keywords

  • Apoptosis
  • Caspase
  • Fas-ligand
  • Mitochondria
  • Permeability transition pore

ASJC Scopus subject areas

  • Toxicology

Cite this

Bcl-2 does not inhibit the permeability transition pore in mouse liver mitochondria. / Yang, Joy C.; Kahn, Axel; Cortopassi, Gino A.

In: Toxicology, Vol. 151, No. 1-3, 26.10.2000, p. 65-72.

Research output: Contribution to journalArticle

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abstract = "The mechanism by which the mitochondrially-localized Bcl-2 protein inhibits apoptosis is still unclear. Some authors have proposed that apoptosis is dependent on induction of the mitochondrial permeability transition pore (PTP), and that activators of apoptosis such as Bax work through activation of PTP, whereas inhibitors of apoptosis such as Bcl-2 work through inhibition of PTP, and the consequent activation or inhibition of PTP-dependent release of mitochondrial apoptotic factors, including cytochrome c. PTP opening is classically measured by a light-scattering assay of large-amplitude swelling of rodent liver mitochondria in sucrose media. Thus to test the hypothesis that Bcl-2 inhibits either the PTP or the PTP-dependent release of cytochrome c, the rate and extent of PTP, and PTP-dependent release of cytochrome c were compared in liver mitochondria from control and Bcl-2 transgenic mice. We demonstrated that Bcl-2 protein was expressed to high levels in mitochondria of transgenics versus controls. We confirmed that while control mice undergo massive hepatic cell death upon exposure to anti-Fas antibody, the Bcl-2 transgenic livers were resistant, by the criteria of gross morphology, serum enzyme release, and caspase 3 activity. We purified mitochondria from livers of the Bcl-2 transgenics and measured PTP directly by the mitochondrial swelling assay. Purified mitochondria from both transgenics and controls were induced to undergo large-amplitude swelling that was dependent on the classical PTP inducers calcium ion (Ca2+), t-butyl hydroperoxide (tBOOH) and atractyloside (Atr); and as expected, pretreatment of mitochondria with cyclosporin A (CsA) completely abolished mitochondrial swelling. However, there was no difference in the rate or final extent of PTP induction in Bcl-2 overexpressors versus control mitochondria. Furthermore, there was no difference in the PTP dependent release of cytochrome c from Bcl-2 overexpressors versus control mitochondria. Therefore, while we observe a strong inhibition of Fas-dependent apoptosis by Bcl-2 overexpression in mouse liver, we observe no effect of Bcl-2 overexpression on either the rate or extent of mitochondrial PTP, or upon the release of cytochrome c from mitochondria in which the PTP has been induced. The simplest explanation of these results is that Bcl-2 inhibits neither PTP nor PTP-dependent release of cytochrome c, however, other possibilities are discussed. Copyright (C) 2000 Elsevier Science Ireland Ltd.",
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AU - Cortopassi, Gino A

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AB - The mechanism by which the mitochondrially-localized Bcl-2 protein inhibits apoptosis is still unclear. Some authors have proposed that apoptosis is dependent on induction of the mitochondrial permeability transition pore (PTP), and that activators of apoptosis such as Bax work through activation of PTP, whereas inhibitors of apoptosis such as Bcl-2 work through inhibition of PTP, and the consequent activation or inhibition of PTP-dependent release of mitochondrial apoptotic factors, including cytochrome c. PTP opening is classically measured by a light-scattering assay of large-amplitude swelling of rodent liver mitochondria in sucrose media. Thus to test the hypothesis that Bcl-2 inhibits either the PTP or the PTP-dependent release of cytochrome c, the rate and extent of PTP, and PTP-dependent release of cytochrome c were compared in liver mitochondria from control and Bcl-2 transgenic mice. We demonstrated that Bcl-2 protein was expressed to high levels in mitochondria of transgenics versus controls. We confirmed that while control mice undergo massive hepatic cell death upon exposure to anti-Fas antibody, the Bcl-2 transgenic livers were resistant, by the criteria of gross morphology, serum enzyme release, and caspase 3 activity. We purified mitochondria from livers of the Bcl-2 transgenics and measured PTP directly by the mitochondrial swelling assay. Purified mitochondria from both transgenics and controls were induced to undergo large-amplitude swelling that was dependent on the classical PTP inducers calcium ion (Ca2+), t-butyl hydroperoxide (tBOOH) and atractyloside (Atr); and as expected, pretreatment of mitochondria with cyclosporin A (CsA) completely abolished mitochondrial swelling. However, there was no difference in the rate or final extent of PTP induction in Bcl-2 overexpressors versus control mitochondria. Furthermore, there was no difference in the PTP dependent release of cytochrome c from Bcl-2 overexpressors versus control mitochondria. Therefore, while we observe a strong inhibition of Fas-dependent apoptosis by Bcl-2 overexpression in mouse liver, we observe no effect of Bcl-2 overexpression on either the rate or extent of mitochondrial PTP, or upon the release of cytochrome c from mitochondria in which the PTP has been induced. The simplest explanation of these results is that Bcl-2 inhibits neither PTP nor PTP-dependent release of cytochrome c, however, other possibilities are discussed. Copyright (C) 2000 Elsevier Science Ireland Ltd.

KW - Apoptosis

KW - Caspase

KW - Fas-ligand

KW - Mitochondria

KW - Permeability transition pore

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