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

The mechanism by which the mitochondrially-localized Bcl-2 protein inhibits apoptosis is still unclear. Some authors have proposed that apoptosis is d ependent on induction of the mitochondrial permeability transition pore (PT P), and that activators of apoptosis such as Bas work through activation of PTP, whereas inhibitors of apoptosis such as Bcl-2, well; through inhibiti on of PTP, and the consequent activation or inhibition of PTP-dependent rel ease of mitochondrial apoptotic factors, including cytochrome c. PTP openin g is classically measured by a light-scattering assay of large-amplitude sw elling of rodent liver mitochondria in sucrose media. Thus to test the hypo thesis that Bcl-2 inhibits either the PTP or the PTP-dependent release of c ytochrome c, the rate and extent of PTP, and PTP-dependent release of cytoc hrome c were compared in liver mitochondria from control and Bcl-2 transgen ic mice. We demonstrated that Bcl-2 protein was expressed to high levels in mitochondria of transgenics versus controls. We confirmed that while contr ol mice undergo massive hepatic cell death upon exposure to anti-Fas antibo dy, the Bcl-2 transgenic livers were resistant, by the criteria of gross mo rphology, serum enzyme release, and caspase 3 activity. We purified mitocho ndria 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 depe ndent on the classical PTP inducers calcium ion (Ca2+), t-butyl hydroperoxi de (tBOOH) and atractyloside (Atr); and as expected, pretreatment of mitoch ondria with cyclosporin A (CsA) completely abolished mitochondrial swelling . However, there was no difference in the rate or final extent of PTP induc tion in Bcl-2 overexpressors versus control mitochondria. Furthermore, ther e was no difference in the PTP dependent release of cytochrome c from Bcl-2 overexpression versus control mitochondria. Therefore, while we observe a strong inhibition of Fas-dependent apoptosis by Bcl-2 overexpression in mou se liver. we observe no effect of Bcl-2 overexpression on either the rate o r extent of mitochondrial PTP, or upon the release of cytochrome c from mit ochondria in which the PTP has been induced. The simplest explanation of th ese results is that Bcl-2 inhibits neither PTP nor PTP-dependent release of cytochrome c, however, other possibilities are discussed. (C) 2000 Elsevie r Science Ireland Ltd. All rights reserved.