Permeability transition in rat liver mitochondria is modulated by the ATP-Mg/Pi carrier
Thilo Hagen,1
Christopher J. Lagace,2
Josephine S. Modica-Napolitano,3 and
June R. Aprille4
1Wolfson Institute for Biomedical Research,
University College London, London WC1E 6BT, United Kingdom;
2Department of Physiology, Tufts University School of
Medicine, Boston 02111; 3Department of Biology,
Merrimack College, North Andover, Massachusetts 01845; and
4Department of Biology, University of Richmond,
Richmond, Virginia 23173
Submitted 30 January 2003
; accepted in final form 27 March 2003
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ABSTRACT
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Mitochondrial permeability transition, due to opening of the permeability
transition pore (PTP), is triggered by Ca2+ in
conjunction with an inducing agent such as phosphate. However, incubation of
rat liver mitochondria in the presence of low micromolar concentrations of
Ca2+ and millimolar concentrations of phosphate is known
to also cause net efflux of matrix adenine nucleotides via the
ATP-Mg/Pi carrier. This raises the possibility that adenine
nucleotide depletion through this mechanism contributes to mitochondrial
permeability transition. Results of this study show that phosphate-induced
opening of the mitochondrial PTP is, at least in part, secondary to depletion
of the intramitochondrial adenine nucleotide content via the
ATP-Mg/Pi carrier. Delaying net adenine nucleotide efflux from
mitochondria also delays the onset of phosphate-induced PTP opening. Moreover,
mitochondria that are depleted of matrix adenine nucleotides via the
ATP-Mg/Pi carrier show highly increased susceptibility to swelling
induced by high Ca2+ concentration, atractyloside, and
the prooxidant tert-butylhydroperoxide. Thus the ATPMg/Pi carrier,
by regulating the matrix adenine nucleotide content, can modulate the
sensitivity of rat liver mitochondria to undergo permeability transition. This
has important implications for hepatocytes under cellular conditions in which
the intramitochondrial adenine nucleotide pool size is depleted, such as in
hypoxia or ischemia, or during reperfusion when the mitochondria are exposed
to increased oxidative stress.
adenine nucleotides; calcium; phosphate; hypoxia; transport
THE PERMEABILITY TRANSITION pore (PTP) in liver mitochondria is
a cyclosporin A-sensitive, high-conductance channel formed at contact sites
between the mitochondrial inner and outer membranes. The PTP is a
multi-protein complex that includes the voltage-dependent anion channel (outer
membrane), the adenine nucleotide tranlocase (ANT; inner membrane), and
cyclophilin-D (mitochondrial matrix). In vitro, PTP opening causes membrane
depolarization as a primary consequence and swelling due to equilibration of
ionic gradients as a secondary consequence. In vivo, the PTP participates in
the regulation of matrix Ca2+, pH, transmembrane
potential, and volume, and there is evidence to suggest that its opening is a
key event in a variety of toxic, hypoxic, and oxidative forms of cell injury
(7). Mitochondrial permeability
transition has been implicated in both apoptotic and necrotic cell death
(8,
14).
In vitro, liver mitochondria undergo permeability transition in response to
various inducers, including Ca2+, Pi, the ANT
inhibitor actractyloside, and prooxidants such as tert-butylhydroperoxide
(tBH) (8). A fact that is often
overlooked, however, is that standard conditions under which mitochondrial
permeability transition is measured (i.e., the presence of a respiratory
substrate, Ca2+ at micromolar concentrations, and
Pi at millimolar concentrations) can cause net efflux of
intramitochondrial adenine nucleotides
(1,
2). This occurs via the
ATP-Mg/Pi carrier
(1,
2), a
Ca2+ activated, atractyloside-insensitive transporter
that catalyzes the divalent electroneutral exchange of ATP-Mg for
Pi (Fig. 1). The
ATP-Mg/Pi carrier in liver mitochondria is distinct from ANT, which
mediates a membrane potential-dependent one-for-one exchange of cytosolic ADP
for matrix ATP (12) and
therefore does not account for net changes in the matrix adenine nucleotide
content. Experimentally, net uptake and loss of adenine nucleotides via the
ATP-Mg/Pi carrier can be controlled by varying the external
concentration of ATP in the presence of Mg2+ and
Pi (5,
6). Incubation of energized rat
liver mitochondria at an external ATP concentration >1 mM results in a net
uptake of ATP; incubation at an external concentration of ATP <1 mM results
in a net loss of matrix adenine nucleotides; and, incubation at an external
concentration of 1 mM ATP results in a near-equilibrium steady state with no
net movement of adenine nucleotides across the inner mitochondrial membrane.
In vivo, modulation of liver intramitochondrial adenine nucleotide content via
the ATP-Mg/Pi carrier plays an important role in the regulating
metabolic activities that have adenine nucleotide-dependent steps localized to
the mitochondrial compartment (e.g., gluconeogenesis, urea synthesis, and
oxidative phosphorylation)
(1).

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Fig. 1. Adenine nucleotide transport in liver mitochondria. The total matrix
adenine nucleotide size (AdNtotal; sum of ATP + ADP + AMP) in liver
mitochondria is regulated by the ATP-Mg/Pi carrier. This
transporter catalyzes an electroneutral exchange of ATP-Mg carrying two
negative charges for divalent phosphate. Because matrix ATP concentration
([ATP]) is in equilibrium with ADP concentration and AMP concentration,
changes in [ATP] due to net movement via the ATP-Mg/PI carrier
result in proportional changes in the concentrations of other adenine
nucleotides. The ATP-Mg/Pi carrier is distinct from mitochondrial
adenine nucleotide tranlocase (ANT), which mediates a membrane
potential-dependent one-for-one exchange of cytosolic ADP for matrix ATP and
therefore does not account for net changes in the matrix adenine nucleotide
content. These adenine nucleotide carriers are further distinguished by
different inhibitor sensitivities. Whereas the ANT is inhibited by
atractyloside (binding from the cytosolic side) and bongkrekic acid (binding
from the matrix side), the ATP-Mg/Pi carrier is insensitive to
these inhibitors but is activated by micromolar Ca2+
concentrations.
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Changes in the matrix adenine nucleotide content (comprised of the sum of
ATP, ADP, and AMP) are also likely to modulate the susceptibility of
mitochondria to undergo permeability transition, because PTP opening has been
shown to be regulated by molecules, including adenine nucleotides that can
interact with ANT (8,
10). Atractyloside, which
inhibits ANT by binding from the cytosolic side and thus brings the carrier
into the so-called c-state (i.e., the binding center faces the
cytosolic side of the inner membrane), induces PTP opening. In contrast,
bongkrekic acid, which also inhibits ANT but binds from the matrix side and
locks the carrier in the m-state, is a potent inhibitor of pore
opening. Moreover, externally added ANT substrates ATP and ADP also inhibit
mitochondrial permeability transition
(8). During periods of hypoxia
and ischemia, mitochondrial permeability transition is believed to be one of
the main factors contributing to cell damage and death
(8). The decrease in cytosolic
ATP that occurs under these conditions, along with a dramatic rise in the
cytosolic Pi concentration due to phosphate hydrolysis of ATP to
ADP and AMP and a rise in cytosolic Ca2+, is assumed to
trigger opening of the PTP (8).
However, an important consideration often overlooked is that a decrease in
cytosolic ATP and concomitant increases in cytosolic Pi and
Ca2+ are also conditions that will favor adenine
nucleotide loss from mitochondria via the Ca2+-activated
ATP-Mg/Pi carrier
(1,
2). In fact, the mitochondrial
adenine nucleotide content has been shown to be severely depleted under
conditions of hypoxia and ischemia
(9,
15,
18). A role for the
ATPMg/Pi carrier in modulating sensitivity of mitochondria to
undergo permeability transition could therefore have important implications
under cellular conditions such as hypoxia and ischemia or during reperfusion
when the mitochondria are exposed to increased oxidative stress.
The purpose of this study was to examine the role of ATP-Mg/Pi
carrier-dependent changes in the matrix adenine nucleotide content in
modulating PTP opening induced by Pi, Ca2+,
atractyloside, and tBH. Results show that Pi-induced opening of the
PTP in liver mitochondria is, at least in part, secondary to depletion of the
intramitochondrial adenine nucleotide content via the ATP-Mg/Pi
carrier. Furthermore, a decreased matrix adenine nucleotide content increases
the susceptibility of mitochondria to PTP opening induced by
Ca2+, atractyloside, and tBH. In addition, changes in
the cytosolic adenine nucleotide composition also appear to contribute to
increased mitochondrial membrane permeability transition. These data suggest a
role for the ATP-Mg/Pi carrier in modulating the sensitivity of
mitochondria to undergo permeability transition under cellular conditions such
as hypoxia, ischemia, or reperfusion.
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MATERIALS AND METHODS
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Isolation of liver mitochondria. Rat liver mitochondria were
isolated by differential centrifugation as described previously
(6).
Mitochondrial swelling measurements. Mitochondrial swelling was
measured as the decrease in light scattering by following optical density at
540 nm (A540) over time. Standard incubation conditions for the
swelling assay were 250 mM sucrose, 10 mM Tris (pH 7.4), 5 mM succinate, and 5
µM CaCl2. Qualitatively similar results were obtained if sucrose
was replaced by 150 mM KCl in the swelling assay. Mitochondria were added at a
final concentration of 0.5 mg protein/ml, and after equilibration for 1 min,
swelling was induced by different agents as indicated in the figure
legends.
Manipulation of the matrix adenine nucleotide content. To adjust
the matrix adenine nucleotide content, mitochondria were incubated for 15 min
at 30°C under the following incubation conditions (in mM): 225 sucrose, 10
Tris (pH 7.4), 10 KCl, 2 KPi, 5 MgCl2, and 5 glutamate
and malate. External ATP was included at specific concentrations that result
in predictable values of the matrix adenine nucleotide content: no ATP to
completely deplete the mitochondria of adenine nucleotides, 0.15 mM ATP to
deplete adenine nucleotides to intermediate levels, 1 mM ATP to maintain the
adenine nucleotide content approximately at the initial level (1215
nmol/mg protein in freshly isolated liver mitochondria), and 2 mM ATP to
overload the mitochondria with adenine nucleotides
(5,
6). After incubation,
mitochondria were centrifuged, washed once in 250 mM sucrose and 10 mM Tris
(pH 7.4), and then used for the swelling experiments.
Adenine nucleotide measurements. ATP, ADP, and AMP concentrations
were determined enzymatically in neutralized PCA-extracts of mitochondria, as
described previously (6).
Measurements of state 3 respiration. Mitochondrial oxygen
consumption was assayed polarographically at 30°C by using a Clark
electrode (Yellow Springs Instruments) as described previously
(4). The medium used for
respiration consisted of (in mM) 225 sucrose, 10 Tris (pH 7.4), 10 KCl, 1
EDTA, 10 KPi, 5 MgCl2, and 5 succinate. To measure
state 3 respiration, mitochondria were incubated at a protein
concentration of 0.5 mg/ml in the presence of 0.2 mM ADP.
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RESULTS
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Pi, in conjunction with Ca2+, is a
well-known inducing agent of PTP opening. Thus the addition of 10 mM
Pi to isolated rat liver mitochondria in the presence of 5 µM
Ca2+ and 5 mM succinate leads to mitochondrial swelling
as detected by a decrease in light scattering
(Fig. 2A). Results
show also that Pi-induced swelling was inhibited in the presence of
the calcium chelator EGTA. Furthermore, Pi-induced swelling was
sensitive to cyclosporin A (Fig.
2B), indicating that it was due to opening of the
mitochondrial PTP. The initial small decrease in light scattering observed
with cyclosporin A is likely to be caused by osmotic swelling as a result of
Pi uptake by the mitochondria through the Pi
/H+ carrier.
However, in addition to inducing PTP opening, millimolar concentrations of
Pi are also known to induce a rapid net loss of adenine nucleotides
in rat liver mitochondria via the atractyloside-insensitive,
Ca2+ activated ATP-Mg/Pi carrier, which
catalyzes exchange of Pi for ATP-Mg
(1,
2). In the absence of external
ATP, matrix ATP-Mg exchanges for external Pi leading to a decrease
in the matrix adenine nucleotide pool. It may be hypothesized, therefore, that
Pi-induced adenine nucleotide loss via the ATP-Mg/Pi
carrier could account, at least in part, for the opening of the PTP on
Pi addition and thus plays a role in increasing the susceptibility
of mitochondria to undergo permeability transition.
Figure 3A
demonstrates the rapid loss of matrix adenine nucleotides in rat liver
mitochondria exposed to 10 mM Pi. No matrix adenine nucleotides
were lost in the absence of Pi or in the presence of Pi
and EGTA; under these conditions the Ca2+-dependent
ATP-Mg/Pi carrier is inactive
(Fig. 3A).
Furthermore, Pi-induced adenine nucleotide loss was delayed if the
mitochondrial ATP synthase inhibitor oligomycin was added before the addition
of the respiratory substrate (succinate). Under these conditions, no ATP can
be synthesized, and therefore, the matrix ATP concentration is very low
relative to the ADP concentration. Thus availability of matrix ATP for
exchange against external Pi via the ATP-Mg/Pi-carrier
is limited. The delay in Pi-induced adenine nucleotide loss in the
presence of oligomycin (Fig.
3A) correlates well with a delay in the onset of
Pi-induced swelling in the presence of oligomycin
(Fig. 3B). This
finding suggests that the initial swelling with Pi was related to a
rapid loss of matrix adenine nucleotides rather than a direct consequence of
Pi on the PTP.

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Fig. 3. Adenine nucleotide depletion is a contributing cause of phosphate-induced
swelling. Mitochondria were incubated under standard conditions in the absence
or presence of 10 mM Pi ± 1 mM EGTA or 1 µg/ml oligomycin
as indicated. A: at various time points between 0 and 20 min, 1 ml
aliquots of mitochondria were removed from the incubation and centrifuged, and
the adenine nucleotide content (ATP + ADP + AMP) was determined enzymatically
in neutralized PCA extracts of the mitochondrial pellets. B:
mitochondrial swelling was measured as A540 over the 20-min
time course.
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To further test whether the matrix adenine nucleotide content affects PTP
opening, mitochondrial adenine nucleotides were depleted by another method,
i.e., by the addition of pyrophosphate. External pyrophosphate exchanges via
the atractyloside-sensitive ANT for matrix ADP
(13), resulting in a rapid
decrease in the matrix adenine nucleotide content.
Figure 4 shows that the onset
of Pi-induced swelling in the presence of oligomycin was
accelerated if 0.5 mM pyrophosphate was added. Pyrophosphate addition had no
effect on the onset of swelling if carboxyatractyloside was present to inhibit
the ADP/ATP carrier, ruling out any unspecific effect of pyrophosphate.

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Fig. 4. Pyrophosphate-induced depletion of matrix adenine nucleotides enhances
mitochondrial swelling. Mitochondrial swelling, measured under standard
conditions, was induced by 10 mM Pi, in the presence or absence of
0.5 mM pyrophosphate (PPi). Oligomycin (1 µg/ml) or
carboxyatractyloside (CA; 5 µM) was added as indicated. NaF (10 mM) was
included in all assays to inhibit pyrophosphate hydrolysis.
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Thus the data presented so far indicate that Pi-induced opening
of the PTP is, at least in part, secondary to depletion of mitochondrial
adenine nucleotides. Results show that loss of adenine nucleotides via either
the ATP-Mg/Pi carrier or the ANT will induce mitochondrial
swelling. However, under physiological conditions, only the
ATP-Mg/Pi carrier is likely to mediate net adenine nucleotide
efflux, because mitochondrial adenine nucleotide loss has been demonstrated in
intact hepatocytes on hormonal stimulation with glucagon or vasopressin, which
increase the intracellular Ca2+ concentration leading to
activation of the ATPMg/Pi carrier
(9). In contrast, the ANT,
which mediates a one-for-one exchange of ATP for ADP, does not contribute to
adenine nucleotide net transport in liver mitochondria
(6). The low intracellular
pyrophosphate concentration and the low affinity of the ANT for pyrophosphate
compared with ATP and ADP (13)
make it unlikely that pyrophosphate-dependent adenine nucleotide net transport
occurs under physiological conditions.
We then tested whether manipulation of the matrix adenine nucleotide
content via the ATP-Mg/Pi carrier plays a general role in
modulating the susceptibility of isolated mitochondria to undergo permeability
transition. For these experiments, the intramitochondrial adenine nucleotide
content (ATP + ADP + AMP) was adjusted to set values between 2.9 and 17.5
nmol/mg mitochondrial protein by preincubating mitochondria with varying
external ATP concentrations between 0 and 1.0 mM at 30°C for 15 min as
described under MATERIALS AND METHODS. Depending on the specified
external ATP concentration, ATP-Mg is released by mitochondria in exchange for
Pi via the ATP-Mg/Pi carrier to a predictable
steady-state level (5,
6). Note that the normal matrix
adenine nucleotide content of freshly isolated rat liver mitochondria is
between 12 and 15 nmol/mg protein.
Figure 5A shows
that under standard incubation conditions (i.e., 5 mM succinate and 5 µM
Ca2+) mitochondria with the lowest adenine nucleotide
content (2.9 nmol/mg protein) underwent modest swelling even in the absence of
any added Pi. This swelling was completely inhibited by cyclosporin
A. In the presence of 0.5 mM Pi and oligomycin to prevent further
adenine nucleotide loss via the ATP-Mg/Pi carrier (oligomycin
prevents phosphorylation of matrix ADP to ATP), both the rate of onset and
magnitude of swelling of severely depleted mitochondria was enhanced
(Fig. 5B). No swelling
was observed in moderately depleted or nondepleted mitochondria in either the
absence or presence of 0.5 mM Pi and oligomycin
(Fig. 5, A and
B).

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Fig. 5. Mitochondrial swelling is enhanced in mitochondria that have a low matrix
adenine nucleotide content. For these experiments, the intramitochondrial
adenine nucleotide content (ATP + ADP + AMP) was manipulated to set values
between 2.9 and 17.5 nmol/mg mitochondrial protein by preincubating
mitochondria with varying external ATP concentrations as described under
MATERIALS AND METHODS. For comparison, the normal matrix adenine
nucleotide content of freshly isolated rat liver mitochondria is between 12
and 15 nmol/mg protein. Mitochondrial swelling was measured under standard
incubation conditions in the absence (A) or presence (B) of
0.5 mM Pi and 1 µg/ml oligomycin. Cyclosporin A (1 µM) was
included as indicated.
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Figure 6 compares the
effects of Ca2+, tBH or atractyloside on swelling in
mitochondria that were moderately depleted of adenine nucleotides and in
nondepleted mitochondria, which do not undergo rapid swelling in the absence
of inducing agents. In the absence of Pi, 150 µM
Ca2+ induced rapid swelling in mitochondria that were
depleted to an adenine nucleotide content of 5.48 nmol/mg protein, whereas in
nondepleted mitochondria, almost no swelling was observed
(Fig. 6A).
Ca2+-induced swelling in adenine nucleotide-depleted
mitochondria was completely prevented with 1 µM cyclosporin A or 2 µM
ruthenium red (not shown). Mitochondria moderately depleted of adenine
nucleotides also showed increased sensitivity to swelling induced by 1 mM tBH
(Fig. 6B) that was
fully inhibited in the presence of 1 µM cyclosporin A (not shown). Finally,
increased susceptibility to undergo PTP opening was observed as well in
adenine nucleotide-depleted mitochondria when swelling was induced with 50
µM atractyloside (Fig.
6C). Atractyloside-induced swelling was completely
prevented in the presence of 1 µM cyclosporin A or 10 µM bongkrekic acid
(not shown).

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Fig. 6. Mitochondria moderately depleted of matrix adenine nucleotides show an
increased susceptibility to swelling induced by Ca2+,
tert-butylhydroperoxide (tBH), and atractyloside. In the absence of
Pi, 150 µM Ca2+ induced rapid swelling in
mitochondria moderately depleted of adenine nucleotides (to 5.48 nmol/mg
protein), whereas in nondepleted mitochondria, almost no swelling was observed
(A). Mitochondria moderately depleted of adenine nucleotides also
showed increased sensitivity to swelling induced by 1 mM tBH (+20 µM
Ca2+) (B), and 50 µM atractyloside (+20
µM Ca2+) (C).
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To determine whether PTP opening in adenine nucleotide-depleted
mitochondria was dependent on the intramitochondrial ATP/ADP ratio,
Ca2+-induced swelling and adenine nucleotide
concentrations were measured in these mitochondria in the presence or absence
of oligomycin. Figure 7 shows
that even a 10-fold difference in ATP/ADP ratios produced no difference in
Ca2+-induced swelling. To be able to compare initial
rates, the swelling was accelerated by including a low concentration of
Pi (0.2 mM) in the incubation. At this concentration, Pi
does not lead to further loss of matrix adenine nucelotides over 5 min
(Fig. 7). The low ATP/ADP ratio
with oligomycin present did not affect the rate of
Ca2+-induced swelling
(Fig. 7), suggesting that in
adenine nucleotide-depleted mitochondria either intramitochondrial ATP or ADP
can inhibit PTP opening. Taken together, Figs.
5,
6,
7 show that it is the total sum
of matrix ATP and ADP and not the relative ATP/ADP ratio that determines the
susceptibility of mitochondria to undergo PTP opening.

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Fig. 7. Effect of the intramitochondrial ATP/ADP ratio on the
Ca2+-induced swelling rate. Swelling of mitochondria
partially depleted of adenine nucleotides was measured under standard
incubation conditions in the presence of 0.2 mM Pi and in the
absence or presence of 1 µg/ml oligomycin. Swelling was induced by the
addition of 50 µM Ca2+. The matrix adenine nucleotide
contents given in the table were measured in the same mitochondrial
preparations under identical incubation conditions. To determine the actual
ATP/ADP ratio under the incubation conditions, instantaneous separation of
mitochondria from the incubation medium was necessary. This was done by rapid
centrifugation of 1 ml aliquots layered over silicone oil and perchloric acid,
as previously described (3).
The perchloric acid extracts of the mitochondrial pellets were neutralized and
adenine nucleotide concentrations were measured enzymatically.
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Results indicate that mitochondria depleted of adenine nucleotides to
levels of 56 nmols/mg protein or below are prone to undergo
permeability transition, which could be of significance under conditions of
hypoxia and ischemia when matrix adenine nucleotides are low. Conditions of
prolonged hypoxia and anoxia have also been shown to lead to a marked decrease
in cytosolic ATP and ADP and a concomitant increase in AMP in hepatocytes
(9). Therefore, we were
interested in measuring swelling in the presence of external ATP, ADP, or AMP
in mitochondria moderately depleted of adenine nucleotides. Either ATP or ADP
at an external concentration of 1 mM completely prevented swelling induced by
Ca2+, tBH, or atractyloside
(Fig. 8,
AC). External 1 mM AMP was less effective
in inhibiting Ca2+-induced swelling
(Fig. 8A).
Interestingly, 1 mM AMP actually accelerated the onset of swelling induced
with tBH or atractyloside (Fig. 8,
B and C). These results indicate that not only
depletion of the matrix adenine nucleotide pool, but also changes in the
composition of adenine nucleotides in the cytosol, may modulate the
susceptibility of mitochondria to undergo permeability transition.

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Fig. 8. Effect of external ATP, ADP, and AMP on mitochondrial swelling induced by
Ca2+, tBH, and atractyloside. Swelling in mitochondria
depleted of adenine nucleotides to 5.85 nmol/mg protein was induced by 150
µM Ca2+ (A), 1 mM tBH (+20 µM
Ca2+) (B), or 50 µM atractyloside (+20 µM
Ca2+) (C) in the presence and absence of
externally added 1 mM ATP, ADP, or AMP as indicated.
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So far the results suggest a direct effect of matrix adenine nucleotide
content on permeability transition, probably through occupancy of ANT binding
sites at the PTP on the matrix face by either ATP or ADP. Additional
experiments suggested that adenine nucleotide loss may also have an indirect
effect on mitochondrial permeability transition.
Figure 9 shows that
mitochondrial state 3 respiration varies as a function of matrix
adenine nucleotide content, as has been reported previously
(1,
4). Therefore, the low matrix
adenine nucleotide content that occurs under conditions of tissue hypoxia and
ischemia can be expected to lead to decreased oxidative phosphorylation. This
contributes to a decrease in cytosolic ATP and ADP concentrations and an
increase in AMP concentration and consequently leads to an increase in
susceptibility for mitochondrial permeability transition, possibly through
decreased binding of ATP and ADP to the cytosolic face of ANT in the PTP.

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Fig. 9. Mitochondrial state 3 respiration as a function of the matrix
adenine nucleotide content. The mitochondrial adenine nucleotide content was
manipulated in preincubation to values between 2.67 and 21.98 nmol/mg protein
and state 3 respiration (in the presence of 0.2 mM ADP) measured for
each preparation as described under MATERIALS AND METHODS.
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DISCUSSION
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Several important findings are revealed by this study. First, it is
demonstrated that Pi-induced opening of the PTP in isolated
mitochondria is due in some measure to depletion of the intramitochondrial
adenine nucleotide pool via the ATP-Mg/Pi carrier. This result
questions the general assumption that Pi exerts its effect on
mitochondrial permeability transition solely through direct effects on the PTP
complex, because we have shown that the effect of Pi may be largely
secondary to the Pi-induced efflux of matrix ATP via the
ATP-Mg/Pi carrier.
Second, it is shown that a decrease in the intramitochondrial adenine
nucleotide pool leads to increased susceptibility for PTP opening induced by
various inducing agents. These results should be taken into account when
measuring the PTP opening under conditions that favor adenine nucleotide loss.
These findings may also be of significance in intact cells under conditions of
hypoxia and ischemia in which the matrix adenine nucleotide content is known
to be decreased. In this situation, there is greater likelihood of the PTP
opening being induced by elevated cytosolic Ca2+
concentration or increased oxidative stress during reperfusion. The
ATP-Mg/Pi carrier, by mediating a loss of mitochondrial adenine
nucleotides to the cytosol under these conditions, thus plays an important
role in the sequence of events leading to cellular dysfunction and
apoptosis.
Third, our study confirms previous reports that external ATP or ADP is also
protective against mitochondrial permeability transition
(10,
16). The effects of
intramitochondrial ATP and ADP as well as externally added ATP and ADP on
permeability transition we observed are likely mediated via their binding to
the ANT, a component of the PTP complex. This hypothesis is supported by the
demonstration that carboxyatractyloside antagonizes the inhibitory effect of
ADP on pore opening (10) and
that reconstituted purified ANT exhibits Ca2+-dependent
channel activity inhibited by ADP
(17). Our results agree with
previous reports (10) that
external AMP is unable to inhibit PTP opening, and we found that AMP even
accelerates permeability transition induced by tBH and atractyloside. The lack
of inhibition of the PTP by AMP may be due to its inability to interact with
the ANT (11).
In conclusion, the results demonstrate that both the intramitochondrial and
the cytosolic adenine nucleotide concentration and composition dynamically
regulate permeability transition. A moderate decrease in the matrix adenine
nucleotide content, as observed under conditions of transient hypoxia or
ischemia, without complete dephosphorylation of cytosolic adenine nucleotides,
should be readily reversible for two reasons: first, because mitochondrial
permeability transition is inhibited by cytosolic ATP and ADP, and second,
because reuptake of adenine nucleotides by the mitochondria can occur on
reoxygenation if cytosolic ATP is available as a substrate for the
ATP-Mg/Pi carrier. Severe or prolonged hypoxic/ischemic stress
ultimately leads to a dramatic decrease in cytosolic ATP and ADP and a
concomitant increase in AMP. As a result, uncontrolled mitochondrial
permeability transition will be more likely, leading to mitochondrial
depolarization and release of proapoptotic proteins, e.g., cytochrome
c and apoptosis-inducing factor as well as to complete depletion of
matrix adenine nucleotides. Dephosphorylation of cytosolic adenine nucleotides
also precludes reuptake of adenine nucleotides by the mitochondria via the
ATP-Mg/Pi carrier on reoxygenation/reperfusion, making these
changes irreversible. It is thus postulated that via modulation of
mitochondrial permeability transition, changes in matrix and cytosolic adenine
nucleotide concentrations play a role in hepatocyte adaptation to and recovery
from transient hypoxia, as well as in cellular dysfunction under conditions of
extreme hypoxia and ischemia that can ultimately lead to apoptotic or necrotic
cell death. The ATP-Mg/Pi carrier, when activated by elevated
cytosolic Ca2+, mediates the shift of adenine
nucleotides from mitochondria to the cytosol and may therefore play an
important role in the regulation of mitochondrial permeability transition
under conditions of hypoxia and ischemia in hepatocytes.
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DISCLOSURES
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J. Aprille was at Tufts University when this work was done and the support
received there in the Department of Biology for this study is gratefully
acknowledged.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. R. Aprille, Dept. of
Biology, University of Richmond, Richmond VA 23173.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
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