Genetics of Apoptosis

2.7

Proapoptotic functions: different designs

What conclusions can be drawn from the inconsistent results cited above?
Mitochondrial responses, such as permeability transition and swelling, are influenced
by the mitochondrial suspension media, the energization state of mitochondria, and
even the specific redox donor employed. In Table 2, assay conditions for addition of
recombinant Bax protein to isolated mitochondria are listed. Methodological
differences in osmotic strength (sucrose/mannitol vs. KCl), divalent cations (Mg2+
vs. Ca2+), adenine nucleotide (+/- regenerating system), and redox donor (succinate
vs. glutamate/malate) have yet to be compared systematically for Bax-mediated
cytochrome c release (for example, the requirement for Ca2+ in Narita et al.; 1998).
Additional variables are likely to modify the membrane insertion and topology of
Bax. As demonstrated by Pastorino et al.; 1999, mitochondrial swelling and
depolarization are dependent on Bax concentration. Bax concentration effects may
reflect the operation of more than one mechanism (such as PTP activation versus
direct channel formation versus lipidic pore) with CsA-resistant mechanisms
predominant at certain concentration ranges. The study by Eskes et al.; 1998 is the
only one to employ full-length Bax, and shows a lack of inhibition by the classic PTP
inhibitors CsA and bongkrekic acid. Recent reports showing that BH3-only proteins,
such as Bid, release mitochondrial cytochrome c by a non-PTP mechanism suggest
that re-examining the Bax/PTP relationship in the context of full-length Bax versus
truncated Bax may be worthwhile (Shimizu and Tsujimoto, 2000; Kim et al., 2000b;
Zhai et al., 2001). It is also possible that CsA binding to mitochondrial cyclophilin
D affects certain processes in addition to PTP formation. A recent report by Scorrano
et al. (2002) described tBid-induced ultrastructural changes to intra-mitochondrial
cristae, consistent with opening of junctions between cristae and intermembrane
spaces. This reorganization is associated with mitochondrial release of an additional
cytochrome c pool that resides in the intercristal spaces and is inhibited by CsA. This
CsA-dependent process may thus amplify cytochrome c release initiated by a separate
mechanism (such as direct channel formation).

56 GENETICS OF APOPTOSIS