THEMES
Mechanisms of Hepatic Toxicity
V. Necrapoptosis and the
mitochondrial permeability transition: shared pathways to necrosis
and
apoptosis*
John J.
Lemasters
Department of Cell Biology & Anatomy, University of North
Carolina, Chapel Hill, North Carolina 27799-7090
 |
ABSTRACT |
Opening of a high-conductance pore conducting
solutes of molecular mass <1,500 Da causes onset of the mitochondrial
permeability transition (MPT). Cyclosporin A blocks this pore and
prevents acute necrotic cell death in several models. Confocal
microscopy directly visualizes onset of the MPT during acute
cytotoxicity from the movement of the green-fluorescing fluorophore,
calcein, into the mitochondria from the cytosol. The MPT also plays a
causative role in tumor necrosis factor-
-induced apoptosis in
hepatocytes. Progression to apoptosis or necrosis after the MPT may
depend on the presence or absence, respectively, of ATP. Often,
features of both apoptotic and necrotic cell death develop after death signals and toxic stresses. The term "necrapoptosis" is
introduced to emphasize the shared pathways leading to both forms of
cell death.
ATP; confocal microscopy; cyclosporin A; tumor necrosis
factor-
 |
INTRODUCTION |
UNTIL RECENTLY, THE mitochondrial
permeability transition (MPT) was an obscure phenomenon
associated with mitochondrial swelling and lysis when the organelles
were treated somewhat harshly in vitro. Studied by only a handful of
researchers until the late 1980s, the MPT was first characterized by
Hunter et al. (14) as a reversible
Ca2+-induced permeabilization of
the mitochondrial inner membrane (reviewed in Refs. 2 and 40).
Permeabilization in the MPT is selective for solutes of molecular mass
less than ~1,500 Da. Single-channel recordings subsequently confirmed
that opening of a nonspecific, high-conductance pore in the inner
membrane precipitates the MPT (37). Conductance of this pore is so
great that opening of only a few pores, possibly only one, is
sufficient to cause mitochondrial depolarization, uncoupling of
oxidative phosphorylation, and large-amplitude mitochondrial swelling,
the signature changes of the MPT (40).
The list of agents that promote onset of the MPT is long (see Ref. 10).
Notably, Ca2+, which must be
transported into mitochondria, inorganic phosphate, reactive oxygen
species (ROS), and a variety of oxidant chemicals induce onset of the
MPT. In addition, membrane depolarization and cross-linking of thiols
in the pore complex promote pore conductance (1, 6). Other factors
block onset of the MPT. These include Mg2+ (pH below ~7), a variety of
phospholipase inhibitors (including bibucaine, mepacrine, and
trifluoperazine), and the immunosuppressive cyclic endecapeptide,
cyclosporin A. Indeed, saturable inhibition of the MPT by nanomolar
concentrations of cyclosporin A removed any doubt that the MPT was
caused by opening of a specific pore in the mitochondrial inner
membrane rather than by a less specific perturbation of lipid bilayer organization.
Given the low abundance of the pore, it is not surprising that
molecular characterization of the pore complex has progressed slowly.
Observations that inhibitors of the adenine nucleotide translocator
(ANT) either induce or inhibit the MPT led to the proposal that the ANT
is an essential component of the permeability transition pore (11) and
pore conductance has been reconstituted with purified ANT (5).
Inhibition of pore conductance by cyclosporin A suggests that the
cyclosporin A binding protein, cyclophilin D, found in the
mitochondrial matrix is also a component of the pore complex. Other
evidence suggests that the pore complex contains VDAC (voltage-gated
anion channel), a protein in the outer membrane. These various
components presumably come together at contact sites between the inner
and outer membranes. Several partially purified preparations with pore
conductances have been described that contain additional proteins with
possible regulatory effects, including hexokinase, creatine kinase, and
the proapoptotic protein, Bax (3, 26). However,
not all evidence supports this model. One report claims to measure pore
conductance from mitochondrial membranes of triple ANT knockout yeast
strains, implying that the ANT is not an obligatory component of the
pore complex (24), and another proposal is that the permeability
transition pore is part of the import machinery that translocates
nucleus-encoded proteins into mitochondria (23).
 |
MPT IN ACUTE CELL INJURY |
In 1990, soon after the discovery that cyclosporin A inhibits the MPT,
the first report appeared that cyclosporin A blocks cell death after an
injurious stress (15). Subsequently, a large number of reports showed
cytoprotection by cyclosporin A against injury from oxidative stress,
anoxia, ischemia-reperfusion, and a variety of toxic chemicals
(reviewed in Ref. 21). However, cyclosporin A has other pharmacological
effects. Its immunosuppressive action, which is independent of its
effect on the MPT, acts by inhibition of calcineurin, a protein
phosphatase involved in T cell activation (12). Thus calcineurin
inhibition might be the basis for cytoprotection. Moreover, free
Mg2+ in the cytosol of normal
cells is 0.5 mM or greater, a concentration that strongly inhibits the
MPT in isolated mitochondria. Therefore, it remained possible that
cytoprotection by cyclosporin might be unrelated to the MPT, and in
situ documentation of onset of a cyclosporin A-sensitive MPT in cells
during the progression of injury was necessary.
Direct observation of the MPT in situ became possible with use of the
three-dimensional resolving power of laser scanning confocal microscopy
(31). When calcein, a green-fluorescing dye, was ester loaded into the
cytosol of cultured cells such as rat hepatocytes and rabbit cardiac
myocytes, confocal microscopy revealed numerous dark round voids in the
otherwise diffuse green fluorescence of the cytoplasm (Fig.
1). Each of these voids is a single
mitochondrion, and the voids exist for the simple reason that the
mitochondrial inner membrane is impermeable to calcein, an organic
polyanion of 623 Da. In contrast, after exposure of hepatocytes to
toxic stresses, including oxidant chemicals,
ischemia-reperfusion, Reye-related drugs, and
Ca2+ ionophore, calcein abruptly
redistributes from the cytosol into the mitochondria, causing the dark
round voids to fill with fluorescence (see Ref. 21) (Fig. 1).
Simultaneously, the mitochondria depolarize, as indicated by the
release of membrane potential-indicating dyes like tetramethylrhodamine
methyl ester. Importantly, cyclosporin A and other MPT blockers prevent
increased permeability of mitochondria to calcein and loss of the
mitochondrial membrane potential (Fig. 1). Furthermore, the MPT
blockers prevent onset of cell death, which strongly supports the
hypothesis that the MPT is a causative mechanism in these models of
acute necrotic cell killing.

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Fig. 1.
Confocal microscopy of onset of the cyclosporin A-sensitive
mitochondrial permeability transition (MPT) in hepatocytes exposed to
Ca2+ ionophore. Cultured rat
hepatocytes were loaded with calcein to monitor mitochondrial membrane
permeability. In the basal images, mitochondria were dark round voids
excluding calcein. After addition of 10 µM Br-A-23187, the dark voids
filled with calcein, and fluorescence became uniform within individual
cells (middle and
right of top
row). As cell viability was lost, calcein abruptly
leaked out (top right). In the
presence of 1 µM cyclosporin A (CsA), the mitochondrial MPT did not
occur after Br-A-23187 addition, and mitochondria continued to exclude
calcein (bottom row). Moreover, cell
viability was not lost. Adapted from Ref. 33.
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 |
THE PH PARADOX AND OXIDATIVE INJURY |
During ischemia, tissue pH decreases rapidly. Rather than
aggravating injury, this acidosis delays the onset of cell death. However, restoration of normal pH after reperfusion accelerates cell
killing, a phenomenon called the pH paradox (7). ROS do not cause
pH-dependent cell killing, since anaerobic reperfusion at normal pH
causes as much cell killing as reperfusion in the presence of oxygen.
Although many factors may contribute to pH-dependent reperfusion
injury, onset of the MPT is perhaps the most important one. When anoxic
hepatocytes at pH 6.5 are "reperfused" with normoxic buffer at pH
7.4, the MPT visualized by calcein redistribution occurs as
intracellular pH rises to ~7 (34). Subsequently, the cells lose
viability. If, at the time of reperfusion, the cells are exposed to
cyclosporin A, then calcein does not redistribute from the cytosol into
the mitochondria. Instead, the mitochondria repolarize, and cell
viability is retained. Similarly, reoxygenation with acidic buffer
blocks onset of the MPT, permitting mitochondrial repolarization and
retention of viability. Thus the MPT is the major mechanism promoting
onset of acute necrotic cell killing in this model of
ischemia-reperfusion injury.
The MPT also plays a causative role in cell killing caused by
tert-butyl hydroperoxide
(t-BuOOH), the short chain analog of lipid hydroperoxides. In cultured hepatocytes, onset of the MPT precedes t-BuOOH-induced cell killing,
and both the MPT and cell death are prevented by the MPT blocker,
trifluoperazine (31). Oxidation of mitochondrial pyridine nucleotides
(NADH and NADPH), an increase of mitochondrial matrix free
Ca2+, and generation of
mitochondria ROS all precede development of the MPT (27, 28). Just as
in isolated mitochondria, each of these mitochondrial changes helps to
promote onset of the MPT and subsequent cell death. Measures that delay
or prevent pyridine nucleotide oxidation, the increase of mitochondrial
Ca2+, and mitochondrial ROS
formation also delay or prevent the MPT and subsequent cell death.
Mitochondrial ROS are also formed during excitotoxic injury to neurons,
and several recent reports implicate the MPT as a causative mechanism
in excitotoxicity (29, 36, 38).
 |
APOPTOSIS |
Necrosis and apoptosis have long been viewed as fundamentally different
processes. Necrotic cell death results from acute metabolic disruption
with ATP depletion, ion disregulation, mitochondrial and cellular
swelling, and activation of degradative enzymes. These
processes culminate in rupture of the plasma membrane and loss of
intracellular proteins, metabolites, and ions. In contrast, apoptosis
represents a special form of cellular differentiation that leads to the
orderly resorption of target cells without severe impairment of
cellular metabolism. Specific signals, such as tumor necrosis
factor-
(TNF-
) and Fas ligand, trigger onset of apoptosis through activation of a cascade of cysteine-aspartate proteases called
caspases (reviewed in Ref. 35).
Despite the differences between necrotic and apoptotic cell death, the
MPT also plays a causative role in apoptosis as well. In a cell free
system combining purified nuclei and mitochondria, onset of the MPT
induces release of soluble mitochondrial factors that activate caspases
and initiate apoptotic nuclear changes (17). These factors include an
apoptosis-inducing factor (AIF) and cytochrome
c, the latter a diffusible electron
carrier in the intermembrane space between the mitochondrial inner and
outer membrane (22). Breakage of the outer membrane after MPT-induced mitochondrial swelling is one likely mechanism by which cytochrome c and AIF are released.
 |
MECHANISM OF CYTOCHROME C RELEASE |
Cytochrome c is the best studied of
the proapoptotic factors released by mitochondria. In the cytosol,
cytochrome c binds to
apoptosis-activating factor-1 (20). Additional binding of ATP (or dATP)
then activates caspase 9, which in turn activates caspase 3. Finally,
caspase 3 stimulates the so-called executioner pathway of apoptosis,
leading to poly(ADP-ribose)polymerase (PARP) cleavage,
internucleosomal DNA hydrolysis, cell shrinkage, chromatin margination,
and nuclear lobulation. Caspases are also involved upstream of release
of cytochrome c from mitochondria.
Binding of TNF-
and Fas ligand to their receptors activates caspase
8. Recent evidence indicates that caspase 8 cleaves Bid, a member of
the Bcl2 family of proteins, which then translocates to mitochondria to
induce cytochrome c release (19, 25)
(see Fig. 2).

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Fig. 2.
Scheme of molecular events in tumor necrosis factor-
(TNF- )-induced apoptosis. TNF- binding to its receptor (TNFR)
activates caspase 8 via the adapter proteins, TRADD and FADD. Bid is
cleaved and translocated to the mitochondria. Onset of the MPT leads to
cytochrome c release and its binding
to apoptosis-activating factor-1 (APAF-1) and ATP (not shown), which is
followed by a cascade of caspase 9 and caspase 3 activation, resulting
in apoptotic cell death. Signaling through another adapter protein,
Traf, activates the nuclear transcription factor, NF B, which leads
to antiapoptotic gene expression acting upstream of mitochondria.
Expression of an I B super-repressor, I B-AA, inhibits the
activation of NF B and is permissive for TNF- -induced apoptosis.
Expression of crmA inhibits the upstream caspase 8 and blocks the MPT
after TNF- addition, whereas inhibition of downstream caspase 3 with
DEVD-cho prevents apoptosis but not the onset of the MPT. Expression of
FADD, a truncated FADD, also blocks apoptotic signaling upstream of
the MPT.
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Whether the MPT actually occurs in cellular apoptosis remains
controversial, and some studies claim that cytochrome
c release during apoptosis occurs
without mitochondrial depolarization (16, 39). However, during
apoptosis in hepatocytes induced by TNF-
, onset of the MPT as
directly visualized by confocal microscopy (Fig.
3) precedes cytochrome
c release, activation of caspase 3, PARP cleavage, internucleosomal DNA degradation, and the morphological changes of apoptosis (4). Cyclosporin A prevents the MPT induced by
TNF-
and blocks cytochrome c
release, caspase 3 activation, and apoptosis. As hepatocytes undergo
apoptosis in this model, onset of the MPT occurs progressively through
the mitochondria of each cell, and 4 or more hours pass between onset
of the MPT in the first and last mitochondrion. For this period of
time, polarized mitochondria coexist with depolarized mitochondria that have undergone MPT, which is consistent with reports of cytochrome c release from cells still containing
polarized mitochondria. Another recent study evaluated the release from
mitochondria of a transfected fusion protein of cytochrome
c and green fluorescent protein (GFP)
during staurosporin-induced apoptosis to PC6 pheochromocytoma cells
(13). Cytochrome c-GFP release
accompanied but did not precede mitochondria depolarization, consistent
with the hypothesis that mitochondrial swelling and outer membrane
rupture after the MPT cause cytochrome
c release. Thus the MPT is in the
middle of the apoptotic signaling cascade, downstream of receptor
binding, caspase 8 activation, and Bid cleavage and upstream of
cytochrome c release and the
activation of caspases 3 and 9 (Fig. 2).

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Fig. 3.
Onset of the MPT during TNF- -induced apoptosis. I B-AA-expressing
cultured rat hepatocytes were treated with TNF- (30 ng/ml) and then
loaded with red-fluorescing tetramethylrhodamine methyl ester (TMRM)
(top row) to monitor mitochondrial
depolarization and green-fluorescing calcein (bottom
row) to monitor the MPT. Between 7 and 12 h of
TNF- treatment, note the loss of mitochondrial TMRM fluorescence and
filling of dark mitochondrial voids with calcein fluorescence,
indicating depolarization and increased mitochondrial
permeability, respectively. After 12 h, note apoptotic shrinkage
and blebbing of one of the cells in the calcein image. Adapted from
Ref. 4.
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ROLE OF ATP IN DIRECTING APOPTOTIC AND NECROTIC CELL KILLING |
If the MPT causes both necrosis and apoptosis, what factor determines
how a cell will die? Apoptosis requires ATP, both in cells and
cell-free systems (8, 18, 22), whereas intracellular ATP actually
prevents onset of necrotic cell death (30). Thus the effect of the MPT
on ATP may determine whether necrotic cell death or apoptosis ensues
(Fig. 4). In hepatocytes, rapid onset of
the MPT after Ca2+ ionophore
treatment leads to profound ATP depletion and necrotic cell death
within 45 min. However, if ATP levels are maintained by a glycolytic
substrate such as fructose in the presence of oligomycin to block ATP
hydrolysis by the uncoupler-stimulated mitochondrial ATPase, this
necrotic killing is prevented. Nonetheless, the MPT still occurs, and
apoptosis develops several hours later, which is nearly completely
inhibited by cyclosporin A (33). Thus the MPT is mediating both
necrosis and apoptosis. When the MPT depletes ATP, necrotic cell death
results, but, when the MPT occurs without severe ATP depletion,
apoptosis develops instead (Fig. 4). Presumably, if ATP depletion
develops during the progression of apoptosis, necrotic cell death will
intervene to produce the secondary necrosis that is so often associated
with apoptosis.
 |
NECRAPOPTOSIS |
The MPT is a pathophysiological mechanism shared by both apoptosis and
necrosis. As a consequence, the long-held distinction between apoptotic
and necrotic cell death becomes blurred. Indeed, in tissue injury due
to ischemia-reperfusion, toxic chemicals, and viral infection,
apoptotic and necrotic features often coexist. This has led to
controversies as to whether the apparent apoptosis of acute chemical
toxicity and reperfusion injury is really necrosis in disguise (9, 32).
Presumably, one might also argue that the apparent necrosis in acute
viral disease is actually apoptosis.
Controversies among scientists are generally resolved in one of two
ways: 1) nobody is right and
2) everyone is right. In regard to
the issue of whether apoptosis or necrosis is the predominant mode of
cell death in chemical toxicity and ischemia-reperfusion injury, the facts support the second resolution. Features
characteristic of necrotic and apoptotic cell death are not only
occurring in the same tissues but simultaneously in the same cells.
Unfortunately, implicit in our nomenclature is the assumption that
either one or the other form of cell killing must occur. Hence, a new
term such as "necrapoptosis" is needed. By necrapoptosis, I mean
a process that begins with a common death signal or toxic stress but
that culminates in either cell lysis (necrotic cell death) or
programmed cellular resorption (apoptosis), depending on other modifying factors. Cell death mediated by the MPT illustrates this
idea. When onset of the MPT is rapid and cellular ATP levels drop
dramatically, then early cell lysis ensues. If progression of the MPT
is slower, or if other sources of ATP generation are available, then
profound ATP depletion is avoided, allowing apoptotic signaling to
proceed. Later if ATP levels finally collapse, cell lysis supervenes in
a pattern of secondary necrosis (Fig. 4). Pure apoptosis and pure
necrosis represent extremes in the spectrum of necrapoptotic responses,
but the more typical response of tissues and cells to injurious
stresses and other death signals is a mixture of events associated with
apoptotic and necrotic cell death.
 |
ACKNOWLEDGEMENTS |
This work was supported, in part, by Grants DK-37034 and AG-07218 from
the National Institutes of Health and by the Office of Naval Research.
 |
FOOTNOTES |
*
Fifth in a series of invited articles on Mechanisms of
Hepatic Toxicity.
Address for reprint requests: J. J. Lemasters, Dept. of Cell Biology & Anatomy, Univ. of North Carolina, CB# 7090, 236 Taylor Hall, Chapel
Hill, NC 27799-7090.
 |
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