cooperate with other mitochondrial apoptotic signals to induce transient opening of
the PTP, facilitating release of IMS apoptotic agents into the cytosol, followed by
resealing of the PTP and regeneration of ∆m, allowing continued production of
ATP for the execution phase of apoptosis (Szalai et al., 1999).
How IP 3 R/RyR Ca2+-release channels are activated during apoptosis, and whether
these receptors actually induce Ca2+ spikes that are received by mitochondria during
physiologic forms of apoptosis are questions that remain to be answered. Nonetheless,
several other independent studies suggest that Ca2+ signals between the ER and
mitochondria are important in diverse cell-death pathways. For instance, apoptosis
induced by staurosporin in neural cells (Kruman and Mattson, 1999), C6 ceramide
in U937 cells (Quillet-Mary et al., 1997), glucocorticoid stimulation of lymphocytes,
activation-induced death of T-cell hybridomas, and TNF-induced death of U937cells
(Zamzami et al., 1995) are all blocked by inhibition of mitochondrial Ca2+ uptake.
In addition, changes in ER Ca2+ homeostasis have been shown to affect the sensitivity
of cells to apoptosis. Stable overexpression of calreticulin, a major Ca2+-binding ER
chaperone, increases the ER Ca2+ storage and enhances agonist-induced IP 3 R Ca2+
release, resulting in increased Cyt.c release, caspase activation, annexin V staining,
and tunnel reactivity in response to thapsigargin, staurosporin, and etoposide
(Nakamura et al., 2000). In contrast, calreticulin-null MEFs are defective in IP 3 R-
dependent Ca2+ efflux (Mesaeli et al., 1999), and resist etoposide-, staurosporin-, and
UVB-induced apoptosis, showing decreased Cyt.c release and caspase activation
(Nakamura et al., 2000). Moreover, overexpression of Bcl-2 alters ER Ca2+
homeostasis, and Bcl-2 targeted exclusively to the ER protects cells against many
forms of apoptosis (see below). Therefore, IP 3 R/RyR-mediated spikes in [Ca2+]m may
participate in the mitochondrial phase of apoptosis in many systems. It is important
to emphasize, however, that at present the data suggest that ER Ca2+ signals sensitize
mitochondria to respond to other apoptotic effectors in certain pathways rather than
functioning as an obligate initiation event.
The involvement of the PTP in the permeabilization of the OMM is very
controversial. Many groups have observed, both in isolated mitochondria and in
intact cells, Cyt.c release in the absence of gross mitochondrial swelling or dissipation
of ∆m, suggesting a PTP-independent mechanism for Cyt.c release (Zamzami and
Kroemer, 2001). BAX can homooligomerize in synthetic liposomes, allowing passage
of Cyt.c, and it has been proposed that BH3-only molecules induce the
oligomerization of BAX and BAK in the OMM, forming Cytc-release channels that
act independently of PTP opening (Korsmeyer et al., 2000). Thus, it is possible that
both PTP-dependent and PTP-independent modes of OMM permeabilization exist.
However, recent work by Scorrano et al. (2002) supports an alternative model for
Cyt.c release and OMM permeabilization that involves both opening of the PTP and
formation of specific BAX and BAK channels. Normally, Cyt.c is sequestered in
regions of the IMS specialized for oxidative respiration and characterized by tight
cristae junctions, raising the question of how Cyt.c becomes available for passage
across the OMM in the absence of mitochondrial swelling and cristae unfolding
during apoptosis. Using isolated mitochondria from wild-type or BAX/BAK-deficient
110 GENETICS OF APOPTOSIS