From the
University of Maryland School of
Medicine, Department of Anesthesiology, Baltimore, Maryland 21201 and
Weill Medical College, Cornell University,
Department of Neurology, New York, New York 10021
Received for publication, April 11, 2003 , and in revised form, May 12, 2003.
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ABSTRACT |
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INTRODUCTION |
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The extent to which mitochondria swell in response to accumulation of large Ca2+ loads also varies considerably with experimental conditions and with mitochondrial tissue type (2527). Brain mitochondria are particularly resistant to Ca2+-induced swelling (25, 28); moreover, they represent many cell populations, which may explain their heterogeneous response to large Ca2+ loads and to inhibitors of the PTP, e.g. cyclosporin A (26, 29).
In this study we report that 2-APB (30) prevents Ca2+-induced PTP in non-synaptosomal brain mitochondria in the presence of physiological concentrations of ATP and Mg2+. 2-APB is reported to inhibit Ca2+ efflux from mitochondria in situ in Jurkat T cells following stimulation of cellular Ca2+ influx through activation of capacitative Ca2+ entry (31). Under these conditions, mitochondria transiently accumulate Ca2+, followed by release upon restoration of basal cytosolic Ca2+ levels. The investigators hypothesized that the inhibition of mitochondrial Ca2+ efflux by 2-APB is because of inhibition of the mitochondrial Na+/Ca2+ exchanger (32). Our results do not provide evidence for 2-APB inhibition of the mitochondrial Na+/Ca2+ exchanger but do indicate that mCICR via the PTP is effectively inhibited. This effect of 2-APB on isolated non-synaptosomal brain mitochondria is observed both in the absence of adenine nucleotides, where inhibition of Ca2+-induced swelling is apparent, and in the presence of millimolar concentrations of ATP and magnesium, where swelling is not observed but where Ca2+ induces release of matrix metabolites and cytochrome c. In the presence of ATP, 2-APB-inhibitable Ca2+-induced mitochondrial alterations are not affected by most other PTP inhibitors, including cyclosporin A.
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EXPERIMENTAL PROCEDURES |
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Mitochondrial Ca2+
UptakeMitochondrial-dependent removal of medium
Ca2+ was followed using the impermeant pentapotassium
salt of the ratiometric dye Fura 6F (Molecular Probes, Portland, OR, USA).
Fura 6F (250 nM) was added to a medium containing mitochondria
(0.25 mg/ml) and 125 mM KCl, 20 mM Hepes, 2
mM KH2PO4, 1 µM EGTA, 4
mM MgCl2, 3 mM ATP, 5 mM malate,
and 5 mM glutamate with the pH adjusted to 7.08 with KOH. According
to calculations using the Winmaxc software
(34), the free
[Mg2+] under these conditions is 1 mM.
It was necessary to use Suprapur KCl (Merck) to minimize
Ca2+ contamination, thereby also minimizing the amount
of added EGTA necessary to eliminate deleterious effects of background
Ca2+. All experiments were performed at 37 °C.
Fluorescence intensity was measured in a Hitachi F-2500 fluorescence
spectrophotometer (Tokyo, Japan) using 340/380-nm excitation and 510-nm
emission wavelengths. The [Ca2+]in the medium was
calculated using the ratio calibration approach described by Grynkiewicz
et al. (35). The
Kd for Fura 6F was estimated to be 2.47
µM using the calcium calibration buffer kit number 3 (Molecular
Probes). An essentially identical value was obtained using the calcium
calibration buffer kit number 2 with magnesium (Molecular Probes).
Mitochondrial Membrane PotentialMitochondrial membrane potential was qualitatively assessed by TMRE (Molecular Probes) fluorescence intensity measured in a Hitachi F-2500 fluorescence spectrophotometer using 549- and 580-nm wavelengths for excitation and emission, respectively (37 °C). Because of the small Stokes shift, we assessed the effect of light scatter on the fluorescent signal, and it was found to be less than 10% as compared with TMRE fluorescence. 125 nM dye was added to a medium containing mitochondria (0.25 mg/ml) and 125 mM KCl, 20 mM Hepes, 2 mM KH2PO4, 1 µM EGTA, 4 mM MgCl2, 3 mM ATP, 5 mM malate, and 5 mM glutamate with the pH adjusted to 7.08 using KOH.
Oxygen ConsumptionMitochondrial respiration was recorded at 37 °C with a Clark-type oxygen electrode (Hansatech, UK). The incubation medium contained 125 mM KCl, 20 mM Hepes, 2 mM KH2PO4, 1 µM EGTA, 1 mM MgCl2, 0.8 mM ADP, 5 mM malate, and 5 mM glutamate (or 5 mM succinate plus 1 µM rotenone) with the pH adjusted to 7.08 with KOH. In some experiments as indicated 5 mM succinate (plus 1 µM rotenone) replaced malate plus glutamate as the oxidizable substrate. State 3 (phosphorylating) respiration was initiated by the addition of mitochondria (0.5 mg/ml) to the incubation medium. State 3 respiration was terminated and State 4 (resting) respiration was initiated by the addition of 2 µM oligomycin.
Cytochrome c Release from MitochondriaAliquots of mitochondrial suspensions were taken at specified incubation times, and the mitochondria were separated from the suspending medium by centrifugation at 14,000 x g for 3 min. The supernatant was carefully removed, and both the supernatant and mitochondrial pellet fractions were immediately frozen and stored at 20 °C. Cytochrome c immunoreactivity was quantified in both fractions using an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN). Before measurement, the supernatant and pellet samples were diluted 1:40 and 1:80, respectively. The release of cytochrome c from mitochondria is expressed as the content of cytochrome c in the supernatant as a percentage of the total content of cytochrome c present in the supernatant plus pellet.
NAD+ + NADH Release from MitochondriaAliquots of mitochondrial suspensions were taken at specified incubation times, and mitochondria were separated from the suspending medium by centrifugation at 14,000 x g for 3 min. The supernatant was carefully removed, and both the supernatant and mitochondrial pellet fractions were immediately frozen and stored at 20 °C. NAD+ plus NADH extraction from both fractions was performed according to the method described by Klingenberg (36). Extracts were transferred to 2 ml of assay medium maintained at 30 °C containing 0.2 mg of alcohol dehydrogenase (Sigma), 50 mM Tris-HCl, 0.6 M ethanol, 50 mM Na4P2O7·10 H2O, pH 7.8. NADH fluorescence was followed in a Hitachi F-2500 fluorescence spectrophotometer using 340- and 460-nm wavelengths for excitation and emission, respectively.
Mitochondrial SwellingSwelling of isolated mitochondria was assessed by measuring light scatter at 660 nm (37 °C) in a Hitachi F-2500 fluorescence spectrophotometer. Mitochondria were added at a final concentration of 0.25 mg/ml to 2 ml of medium containing 125 mM KCl, 20 mM Hepes, 2 mM KH2PO4, 1 µM EGTA, 1 mM MgCl2, 5 mM malate, and5mM glutamate with the pH adjusted to 7.08 with KOH. At the end of each experiment, the non-selective pore-forming peptide alamethicin (80 µg) was added as a calibration standard to cause maximal swelling.
ReagentsStandard laboratory chemicals were from Sigma. 2-APB (Sigma), CGP 37157 (Calbiochem), cyclosporin A (Sigma), bongkrekic acid (Calbiochem), alamethicin (Sigma), tamoxifen (Sigma), spermine (Sigma), xestospongin C (Calbiochem), inositol 1,4,5-triphosphate (ICN), thapsigargin (Calbiochem), trifluoperazine (Sigma), bromoenol lactone (Sigma), aristolochic acid (Biomol), bovine serum albumin (Sigma), 7-nitroindazole (Calbiochem), 1400W (Calbiochem), ubiquinone 0 (Sigma), butylated hydroxytoluene (Sigma), N-acetylcysteine (Sigma), catalase (Sigma), and superoxide dismutase (Sigma).
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RESULTS |
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Dose-dependent Inhibition of Mitochondrial Ca2+-induced Ca2+ Release by 2-APB in the Presence of ATPAfter a demonstration that 2-APB is effective at inhibiting PTP in the absence of adenine nucleotides, subsequent experiments were performed in the presence of 3 mM ATP to model more physiologically relevant conditions. In the presence of ATP, brain mitochondria completely accumulated two additions of 400 nmol of Ca2+ and almost sequestered the third addition. This net uptake of more than 2000 nmol of Ca2+ mg1 mitochondrial protein was followed by a slow release of Ca2+ back to the medium (Fig. 2, curve a). Mitochondria suspended in the presence of either 50 or 100 µM 2-APB accumulated the entire Ca2+ load (2400 nmol mg1) without releasing Ca2+ during the course of the 20-min experiment (Fig. 2, curves c and d, respectively). The presence of even 10 µM 2-APB resulted in some inhibition of Ca2+-induced Ca2+ release (Fig. 2, curve b).
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Fura 6F fluorescence responds reliably to [Ca2+] in
the range of 0.550 µM (not shown). Each addition of
Ca2+ to the mitochondrial suspension was a total of 200
µM; however, according to Winmaxc software
(34), the calculated free
[Ca2+] was <100 µM in the presence of
Mg2+ and ATP. Therefore, the peak of the calcium signal
before the onset of calcium uptake by mitochondria was underestimated.
However, the method used (manual additions of calcium in a cuvette system) is
limited in the temporal dimension of the millisecond scale, where much of the
calcium uptake takes place, given the enormous avidity of the uniporter
(Vmax > 1200 nmol Ca2+/mg of
protein/min) and/or the rapid mode of uptake
(37). It is to be noted that
even though the upper range of the bulk
[Ca2+]i in cells reported by Fura 2
(or higher Kd derivatives) fluorescence is in the
relatively low micromolar range
(38), free
[Ca2+]i may well reach the 100
µM range in certain microdomains
(39) such as the
perimitochondrial environment
(40),
(41).
Comparison of 2-APB to Cyclosporin A and Bongkrekic AcidAlthough cyclosporin A was effective at inhibiting the PTP in brain mitochondria in the absence of adenine nucleotides (Fig. 1a), it failed to inhibit mCICR in the presence of ATP (Fig. 3, curve b). Bongkrekic acid, another PTP inhibitor, exhibited an ability to inhibit mCICR in the presence of ATP (Fig. 3, curve c) that was very similar to that of 2-APB (Fig. 3, curve d). Because the vehicle for bongkrekic acid is 1 M NH4OH, an equal volume of vehicle was tested and found to have no effect on Ca2+ uptake and retention (not shown). Although 2-APB inhibited mCICR, it did cause a slight reduction in the rate of Ca2+ uptake, particularly after the third addition of Ca2+ (Fig. 3, curve d). The effects of 2-APB on mitochondrial bioenergetics were further analyzed using measurements of both mitochondrial membrane potential and O2 consumption.
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Protection by 2-APB against Ca2+-induced Loss of
Mitochondrial Membrane
PotentialFig. 4
provides a qualitative evaluation of after the addition of high
Ca2+ loads in the absence and presence of 2-APB (100
µM) using the fluorescent dye TMRE. In the absence of 2-APB,
TMRE fluorescence stabilized within 2 min after the addition of mitochondria
to the medium (Fig.
4a). The subsequent addition of 400 nmol of
Ca2+ caused an immediate increase in fluorescence,
i.e. decrease in
, as expected because of the collapse of
during rapid Ca2+ influx. After the
100-s period, during which added Ca2+ was
accumulated, the TMRE fluorescence decreased but remained much higher than the
original fluorescence in the absence of Ca2+. A similar
pattern was observed after the second addition of Ca2+.
After the third addition, the TMRE fluorescence failed to return toward base
line and gradually increased for many minutes thereafter. When 2-APB was added
to the mitochondrial suspension (Fig.
4b), TMRE fluorescence increased, indicating some
reduction in
by this compound. However, unlike what was observed
in the absence of 2-APB, the subsequent addition of three pulses of
Ca2+ were followed by a return of TMRE fluorescence
toward, and eventually to the level maintained before these additions. Thus,
at 800 s of incubation,
approached complete depolarization in the
absence of 2-APB but was at least partially retained in its presence. These
observations are consistent with the onset of mCICR in the absence but not in
the presence of 2-APB, also observed at
800 s of incubation. Although
2-APB by itself causes partial mitochondrial depolarization, this effect was
not strong enough to impair net
-dependent
Ca2+ uptake. It must be emphasized that because of the
Nernst equation, data from potentiometric dyes follow a logarithmic, not
linear relationship (42). The
observation that is intended for demonstration on this experiment is that
mitochondria depolarize further upon completion of Ca2+
uptake (Fig. 4a)
unless they are pre-treated with 2-APB
(Fig. 4b). The onset
of depolarization upon high Ca2+ loading (
700 s)
coincides exactly with the onset of Ca2+ release
(Fig. 2, curve a). The
choice of the membrane potential-sensitive probe was critical. Many of the
available probes were tested (safranin O, tetramethylrhodamine methylester
perchlorate (TMRM), rhodamine 123, JC-1), but they all exhibited serious
drawbacks due to the necessity of high calcium loading; i.e. safranin
O reduced maximal calcium uptake capacity considerably, as has also been found
by other laboratories (43),
TMRM and rhodamine 123 were very strong respiratory inhibitors and uncouplers
even at low concentrations (20 nM, not shown), affecting
respiration more than TMRE although the opposite has been observed for rat
heart mitochondria (44), and
JC-1 gave an unsatisfactory signal-to-noise ratio but no suppression of
respiratory control (not shown).
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Inhibition of Ca2+-induced Mitochondrial
Cytochrome c and Pyridine Nucleotide Release by 2-APBRelease of
the mitochondrial intermembrane protein cytochrome c typically
accompanies the osmotic swelling and rupture of the outer membrane evoked by
activation of the PTP
(4547).
Loss of mitochondrial matrix pyridine nucleotides is another measure of the
PTP, distinguishing it from a mechanism of depolarization mediated by
activation of a "low conductance" pore
(48).
Fig. 5 provides the results of
enzyme-linked immunosorbent assay determinations of cytochrome c
released into the medium after the addition of Ca2+ and
a comparison of the effectiveness of 2-APB and cyclosporin A at inhibiting
this release. In the absence of added Ca2+, less than
10% of total mitochondrial cytochrome c was lost to the suspending
medium after 1800 s of incubation. After the addition of 2400 nmol of
Ca2+ mg1 protein, 58%
of total cytochrome c was released at 1800 s. No net release was
observed at 700 s, immediately before net Ca2+ release
was observed (Fig. 3). 2-APB
but not cyclosporin A inhibited Ca2+-induced cytochrome
c release, consistent with the differences in the effects of these
drugs observed on mCICR. The same pattern was observed for release of matrix
pyridine nucleotides (Fig. 6). The appearance of pyridine nucleotides in the medium over that observed in the
absence of Ca2+ occurred after the onset of mCICR and
was inhibited by 2-APB but not cyclosporin A.
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Effects of 2-APB on Mitochondrial Bioenergetics and Relationship to Inhibition of mCICRThe effects of 2-APB on respiration by isolated non-synaptosomal brain mitochondria are described in Table I. 2-APB exhibited a dose-dependent inhibition of State 3 respiration with the NADH-linked oxidizable substrates malate plus glutamate, with significant inhibition observed at a concentration of 10 µM and a 57% inhibition observed at 100 µM. 2-APB similarly inhibited uncoupler (FCCP) stimulated respiration (not shown). State 4 respiration was significantly elevated at 10 µM 2-APB and was approximately twice the control rate at 100 µM. Qualitatively similar results were obtained with mitochondria respiring on succinate in the presence of rotenone (Table I). These results indicate that 2-APB is both a mild respiratory inhibitor and uncoupler at concentrations that protect against activation of the PTP.
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The possibility that 2-APB protection against mCICR is related to its
effects on respiration was tested by measuring mitochondrial
Ca2+ uptake and release in the presence of targeted
respiratory inhibition. When NADH-linked respiration is completely inhibited
by rotenone, active Ca2+ uptake can still be driven by
established via ATP-dependent H+ export catalyzed by
the mitochondrial F0F1-ATPase
(49). As shown in
Fig. 7a, the onset of
mCICR in the presence of rotenone occurred at a smaller
Ca2+ load (800 nmol) than that required in the absence
of rotenone (1200 nmol; Fig.
2). Under this state of complete respiratory inhibition, 100
µM 2-APB still inhibited mCICR
(Fig. 7a, thick
versus thin line), indicating that its moderate inhibition of
O2 consumption is unrelated to its ability to inhibit the PTP.
Ca2+ uptake was slower in the presence of 2-APB, likely
due to its moderate uncoupling effect.
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The possible dependence of 2-APB protection against mCICR on mitochondrial ATP hydrolysis was tested by exposing mitochondria to Ca2+ in the absence of rotenone but in the presence of the ATPase inhibitor oligomycin. Under this condition, mCICR was also activated by 800 nmol of Ca2+ and inhibited by 2-APB (Fig. 7b). Ca2+ influx was significantly slower in the presence of 2-APB, as expected by the inhibition of ATPase-dependent Ca2+ uptake (50) together with the inhibition of respiration by 100 µM 2-APB. The observations that 2-APB is effective at inhibiting mCICR in the absence and the presence of either rotenone or oligomycin indicates that its mechanism is closely related to pore opening and not due to its moderate effects on mitochondrial bioenergetics. In addition to that, moderate uncoupling could prove beneficial to a mitochondrion against noxious stimuli, lowering its ability to form free radicals at a basal level (51). Therefore, FCCP concentration was titrated to confer the same extent of depolarization (30 nM, Fig. 7c, curve a) and activation of state 4 respiration (5 nM, Fig. 7c, curve b), and this range of concentration was tested as a possible protectant against PTP; under these conditions, mCICR was still present.
Inhibition of Mitochondrial Na+/Ca2+ Exchanger Does Not Reproduce the Effect of 2-APBStudies demonstrating 2-APB inhibition of mitochondrial Ca2+ efflux after a capacitative Ca2+ entry event hypothesized the involvement of the mitochondrial Na+/Ca2+ exchanger (32). Pretreatment of isolated brain mitochondria with the inhibitor of the Na+/Ca2+ exchanger CGP 37157 (20 µM) in the absence of Na+ did not inhibit mCICR and actually promoted the release of Ca2+ (Fig. 8, curve a). Moreover, the simultaneous presence of 2-APB (100 µM) and CGP 37157 (20 µM) resulted in much slower Ca2+ uptake and partial antagonism of the protective effect of 2-APB (Fig. 8, curve c; compare with Fig. 3, curve d). When medium K+ was substituted for Na+ at 1040 mM, mCICR was unaffected and was not inhibited by CGP 37157 (not shown). It therefore appears that Na+/Ca2+ exchange activity does not contribute to the mCICR observed in these experiments and is not a target of 2-APB.
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Comparison of 2-APB with Other PTP InhibitorsExperiments identical to that described by Fig. 2 performed in the presence of NADH-linked substrates and ATP were conducted in the presence of other compounds reported to inhibit the PTP or other related cellular activities. Results are expressed as the concentration of medium-free Ca2+ present at 1800 s of mitochondrial incubation and after three additions of 400 nmol of Ca2+. Although the presence of 100 µM 2-APB allowed brain mitochondria to maintain the medium [Ca2+] at <1 µM, no compound other than bongkrekic acid inhibited mCICR to an extent that allowed for maintenance of the medium [Ca2+] at <20 µM. These compounds included phospholipase A2 inhibitors, i.e. trifluoperazine, bromoenol lactone, and aristolochic acid (52), (53, 54), BSA, which binds free fatty acids, antioxidants, i.e. superoxide dismutase, catalase, N-acetylcysteine, butylated hydroxytoluene, ubiquinone 0 (55), inhibitors of nitric-oxide synthesis, i.e. 7-nitroindazole and 1400 W (56), and other agents, e.g. spermine and tamoxifen (57, 58), which inhibit the PTP by other mechanisms. Xestospongin C, a potent inositol 1,4,5-triphosphate receptor blocker (59), was also tested due to the fact that it has been proposed to resemble the active, dimerized form of 2-APB (Ref. 60, but see also Dobrydneva and Blackmore (61)). Because of the documented inhibitory effect of 2-APB on inositol 1,4,5-triphosphate receptors (30) and sarco-endoplasmic reticulum Ca2+ ATPase pump (62), it was prudent to exclude the conception that the increase of Ca2+ in the medium and its inhibition by 2-APB upon high calcium loading was attributed to contaminating microsomes and/or endoplasmic reticulum. The addition of inositol 1,4,5-triphosphate (100 µM) or thapsigargin (2 µM) upon completion of Ca2+ uptake did not cause any acceleration of Ca2+ release (not shown).
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DISCUSSION |
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Experiments were performed to determine whether the effects of 2-APB on
respiration and are related to its PTP inhibitory activity. 2-APB
inhibited mCICR in the presence of the respiratory inhibitor rotenone and in
the presence of the ATP synthase inhibitor oligomycin
(Fig. 7, a and
b, respectively). Moreover, mCICR was not inhibited by
the protonophore FCCP at a concentration that mimics the respiratory
uncoupling and mitochondrial membrane depolarization observed with 100
µM 2-APB (Fig.
7c). Therefore, the moderate effects of 2-APB on
respiration are unrelated to its potent inhibition of the PTP. Although
moderate respiratory inhibition by 2-APB is not responsible for PTP
inhibition, it probably accounts for the slightly slower rate of
Ca2+ uptake observed in the presence of 2-APB in the
presence of ATP (Figs. 2 and
3). In the absence of ATP,
however, 2-APB actually accelerated the rate of mitochondrial net
Ca2+ uptake (Fig.
1b). In the absence of ATP, even small additions of
Ca2+ induce the PTP in a significant fraction of the
mitochondrial population (26),
thereby reducing the number of mitochondria capable of energy-dependent
Ca2+ accumulation. Thus, 2-APB accelerates
Ca2+ uptake in the absence of ATP by maintaining a
larger population of energized mitochondria.
The PTP can assume different conductance states, including that of a low
conductance pore (4), that
causes membrane depolarization but not osmotic lysis unless followed by
transition to a high conductance permeability
(66). Because the mCICR
observed in our experiments was accompanied by release of cytochrome
c and pyridine nucleotides at 800 s of incubation (Figs.
5 and
6, respectively), the high
conductance pore was clearly activated at this time. The additional
observation that
was reduced by Ca2+
additions before release of pyridine nucleotides and cytochrome c
supports the possibility that activation of the low conductance pore preceded
that of the high conductance PTP (Fig.
4a). It is therefore possible that 2-APB inhibits the
transition from the low to high conductance mode
(66). The addition of
ruthenium red (1 µM) or its purified active component Ru360 (165
nM) just before the onset of mCICR induced an abrupt release of
Ca2+, supporting the possibility of an induction of
Ca2+ cycling across the mitochondrial membrane
(18,
67), leading progressively to
opening of the pore throughout the heterogeneous mitochondrial population
(26).
Inhibition of the consequences of PTP activation on brain mitochondria by 2-APB provides a rationale for testing the potential neuroprotective actions of this compound. Despite the mild uncoupling effect of 2-APB, it protected against complete depolarization caused by mitochondrial Ca2+ overload (Fig. 4b) and, therefore, protects against Ca2+-induced impairment of oxidative phosphorylation. 2-APB also prevented the loss of matrix pyridine nucleotides and cytochrome c, both also necessary for respiration and oxidative phosphorylation. Release of cytochrome c can stimulate mitochondrial production reactive oxygen species (68), and loss of pyridine nucleotides and other matrix compounds, e.g. glutathione, can compromise their detoxification. Thus, 2-APB may protect against Ca2+-induced mitochondrial oxidative stress as well as metabolic failure.
The results of this investigation also relate to the contribution of mitochondrial Ca2+ transport to normal cellular Ca2+ homeostasis and signal transduction. Mitochondria within cells may form an electrically coupled network, synchronizing electrical signals generated by the PTP (69). Ca2+ plays a pivotal role in induction and priming of the PTP. Therefore, PTP inhibitors and other agents that modulate mitochondrial Ca2+ handling can be useful tools to elucidate the roles of mitochondria in Ca2+-mediated signal transduction.
In addition to our demonstration that 2-APB is an effective blocker of PTP, this compound is a blocker of store-operated Ca2+ entry (70), a phenomenon recently identified to be centrally positioned among signal transduction and [Ca2+]i homeostasis in both excitable and non-excitable cells (71). Inhibition of SOCE by 2-APB was attributed to impairment of both inositol 1,4,5-triphosphate-mediated Ca2+ release (30) and inhibition of TRP channels (72), (73). However, TRP channels have yet to be unequivocally established as the molecular basis for SOCE (74). 2-APB also inhibits the sarco-endoplasmic reticulum Ca2+ ATPase pump (62) and voltage-dependent K+ channels (75) and blocks gap junctions, possibly by interfering with the docking interactions of two gap junctional hemi-channels (76).
Activation of the mitochondrial PTP may oppose SOCE, based on the fact that SOCE is inhibited by high [Ca2+]i and the observation that abolition of Ca2+ uptake by mitochondria inactivates SOCE (7779). Thus, it is possible that release of mitochondrial Ca2+ stores through activation of the PTP elevate [Ca2+]i, thereby inhibiting SOCE. Interpretation of results obtained from SOCE measurements in the presence of 2-APB is, therefore, potentially complicated by the effect this agent has on mitochondrial Ca2+ handling.
Considering the reported effects of 2-APB on TRP channels, we attempted to identify immunoreactivity of TRP family members (TRPC1, -3, -4, -6) in highly purified mitochondrial subfractions (outer membrane, contact sites, inner membrane) but were unsuccessful (not shown). 2-APB may block the activation machinery of the channels participating in SOCE and not the channels themselves (80), (81). Therefore, 2-APB-sensitive channel-activating proteins similar to those involved in SOCE may be present in mitochondria in the absence of TRP channels. These proteins may interact with one or more of the many mitochondrial cation channels that have been identified electrophysiologically but not adequately characterized (82).
In conclusion, we show that 2-APB inhibits mCICR in isolated non-synaptosomal brain mitochondria in the presence of adenine nucleotides and magnesium. Neither inhibition of the reversal of the uniporter nor blockade of the mitochondrial Na+/Ca2+ exchanger could reproduce the effect of 2-APB. Ca2+ release was accompanied by cyclosporin A-insensitive loss of cytochrome c and pyridine nucleotide release, signifying activation of the "high conductance" pore. These observations taken together with others demonstrating inhibition of SOCE by 2-APB indicate a mitochondrial site of action for this drug in addition to other cell membranes.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Anesthesiology, University of Maryland School of Medicine, 685 W. Baltimore St., MSTF 5.34, Baltimore, MD 21201. Tel.: 410-706-4711; Fax: 410-706-2550; E-mail: gfisk001{at}umaryland.edu.
1 The abbreviations used are: mCICR, mitochondrial
Ca2+-induced Ca2+ release; PTP,
permeability transition pore; 2-APB, 2-aminoethoxydiphenyl borate; TMRE,
tetramethylrhodamine ethyl ester perchlorate; FCCP, carbonyl cyanide
4-(trifluoromethoxy)phenylhydrazone; , mitochondrial membrane
potential; SOCE, store-operated Ca2+ entry; TRP,
transient receptor potential.
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ACKNOWLEDGMENTS |
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REFERENCES |
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