From the Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, CH-8093 Zürich, Switzerland
Received for publication, August 26, 2002, and in revised form, January 22, 2003
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ABSTRACT |
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Mitochondria from transgenic mice, expressing
enzymatically active mitochondrial creatine kinase in liver, were
analyzed for opening of the permeability transition pore in the absence
and presence of creatine kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by creatine or cyclocreatine but not by Most vertebrate cell types express cytosolic as well as
mitochondrial isoforms of the enzyme creatine kinase
(CK).1 CK catalyzes the
reversible transphosphorylation of phosphocreatine (PCr) to ATP. The
findings of distinct subcellular localizations of creatine kinases have
led to the formulation of the PCr circuit concept, proposing that sites
of energy production (mitochondria and glycolysis) are tightly linked
via Cr/PCr shuttling to sites of energy consumption (various cellular
ATPases) (1-6). ATP generated by oxidative phosphorylation in
mitochondria reacts with creatine (Cr) to produce PCr, a reaction
mediated by mitochondrial CK (mtCK) located in the intermembrane and
intercristae space. Phosphocreatine then diffuses to the cytosol to
locally regenerate ATP via cytosolic CK from ADP. Cr produced during
this reaction is shuttled back to the mitochondria for recharging it to
PCr. This energy shuttling system is particularly efficient in tissues
with a very high and fluctuating energy demand like skeletal and
cardiac muscle, as well as in brain and neural tissues (7). Thus, in
addition to the generally accepted temporal energy buffering function, PCr also provides a means to spatially buffer energy reserves (3). This
holds especially true for highly polar cells like spermatozoa where the
diffusion of ADP is the limiting factor (8).
Studies with cultured rat hippocampal neurons have shown recently that
creatine protects against glutamate and In an attempt to define the role of mitochondrial creatine kinase on
MPT, we have shown in an earlier study that isolated liver mitochondria
from transgenic mice containing mtCK did not respond by MPT pore
opening upon treatment with Ca2+ plus atractyloside if Cr
or CyCr were present in the medium (17). In the absence of Cr and CyCr,
however, MPT pore opening could be fully induced by Ca2+
plus atractyloside. On the other hand, in liver mitochondria from
control mice without mtCK, pore opening induced by Ca2+
plus atractyloside was independent on the presence or absence of Cr or
CyCr. In the present study we investigated these effects in more
detail, asking the question of whether inhibition of MPT pore opening
by CK substrates depends on mtCK activity and whether the tight
functional coupling between the CK reaction and oxidative phosphorylation, demonstrated recently to take place in situ
(25), would lead to cycling of mitochondrial adenine nucleotides, thus resulting in net production of phosphorylated CK substrates. Our results indicate that substrate channeling between mtCK and adenine nucleotide translocase (ANT) takes place in a tight functionally coupled microcompartment that seems absolutely required for protection of MPT pore opening by creatine.
Source and Preparation of Mitochondria--
Transgenic mice
expressing the ubiquitous mitochondrial creatine kinase isoform in
their liver mitochondria (transgenic liver-mtCK mice) were kindly
provided by Dr. Alan P. Koretsky (National Institutes of
Health, Bethesda). Mice were killed, and the liver was quickly removed
and placed in ice-cold isolation buffer for mitochondria (10 mM Tris-HCl, pH 7.4, 250 mM sucrose)
supplemented with 1 mM EDTA and 0.1% bovine serum albumin.
Livers were homogenized, and the homogenate was centrifuged at 700 × g for 10 min. The supernatant was filtered through two
layers of nylon gauze and centrifuged at 7000 × g for
10 min. The mitochondrial pellet was washed twice with isolation buffer
(without EDTA and bovine serum albumin), centrifuged, and kept on ice.
Respiration and Swelling Measurements--
Mitochondrial oxygen
consumption was measured with a Cyclobios oxygraph (Anton Paar,
Innsbruck, Austria) at 25 °C. The standard medium (2 ml) consisted
of 10 mM Tris/Mops, pH 7.4, 250 mM sucrose, 10 mM phosphate/Tris, 2 mM MgCl2, 0.5 mM EGTA/Tris, 2 µM rotenone, and 50 µM Ap5A. State 4 respiration was stimulated
by addition of 5 mM succinate/Tris. Creatine kinase
substrates (Cr, CyCr, and GPA) were present at 10 mM
concentration, and state 3 respiration was induced by adding 1 mM ATP. Mitochondrial protein concentration in all assays
was 0.5 mg/ml. Deviations from these conditions are specified in the
figure legends.
Mitochondrial MPT pore opening was measured by the convenient swelling
assay and carried out in a UV4 UV/Vis spectrometer (Unicam) connected
to a computer. Swelling curves were recorded at 540 nm, and data points
were acquired every 1/8 s using Vision© software (version
3.10, Unicam Ltd.). Cuvettes containing the mitochondrial suspension
were thermostated to 25 °C. Incubation conditions and induction of
MPT are indicated in the legends to the figures.
Measurement of PCr Production by Quantitative Thin Layer
Chromatography--
Mitochondria were incubated under different
conditions (see legend to Fig. 4) in the presence of
32Pi. Reactions were stopped after defined time
intervals by addition of 1% (final concentration) SDS and centrifuged,
and the supernatants were applied to Silica Gel 60 thin layer plates
(Merck). Running solvent was a mixture of isopropyl alcohol,
ethanol, and 25% ammonia (6:1:3, by volume). Thin layer plates were
air-dried, exposed to a Kodak storage phosphorscreen SO230, and
analyzed with a PhosphorImager (Storm 820, Amersham Biosciences). The
position of PCr in thin layer chromatograms was identified in control
experiments using recombinant brain-type BB-CK incubated in the
presence of radioactive [14C]creatine and cold ATP.
Binding of Exogenously Added CK to Mitochondria--
Mouse liver
mitochondria (0.5 mg/ml) were incubated in standard medium at pH 7.4 with 5 mM glutamate and 2.5 mM malate and without rotenone. Specified amounts of recombinant brain-type BB-CK or
ubiquitous human mtCK (prepared as described elsewhere (26, 27)) were
added. After a 5-min incubation, an aliquot of the suspension was
removed (to measure total CK activity). The rest was centrifuged (5 min
at 7000 × g) to separate mitochondria. CK activity was
measured separately in the supernatants and mitochondrial pellets by a
coupled enzymatic assay (28).
Other Methods--
Protein concentrations were determined by
Bradford assay (Bio-Rad) using bovine serum albumin as a standard.
Mitochondrial adenine nucleotide content was measured by reversed phase
chromatography of acid extracts according to Kay et al.
(25).
To correlate the effects of the different creatine kinase
substrates on MPT pore opening with their ability to stimulate
oxidative phosphorylation, we first measured stimulation of state 3 respiration by ATP and CK substrates in mitochondria oxidizing
succinate. The data of these experiments are summarized in Table
I. In mtCK containing mitochondria from
transgenic mice, substantial stimulation (about 3-fold over state 4)
was observed only with Cr and CyCr (both 10 mM), but not
with GPA, in the presence of externally added ATP, due to endogenous
production of ADP by mitochondrial CK. The absence of a detectable
stimulation with 10 mM GPA agrees with the inability of
mitochondrial CK to phosphorylate this creatine analog (29, 30). No Cr-
or CyCr-stimulated respiration was seen with mitochondria from control
mice, which do not express mtCK in liver (17). These data confirm
earlier findings with the same substrates given to heart mitochondria
(30). In general, with respect to creatine-stimulated respiration,
mitochondria from transgenic liver-mtCK mice are behaving comparably to
mtCK containing heart mitochondria (see also Ref. 17).
-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extramitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was
studied in the presence and absence of creatine. If mitochondrial creatine kinase activity decreased (i.e. by omitting
magnesium from the medium), protection of permeability transition pore
opening by creatine or cyclocreatine was no longer seen. Likewise, when creatine kinase was added externally to liver mitochondria from wild-type mice that do not express mitochondrial creatine kinase in
liver, no protective effect on pore opening by creatine and its analog
was observed. All these findings indicate that mitochondrial creatine kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate mitochondrial permeability transition in the
presence of creatine. These results are of relevance for the design of creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid toxicity (9), as
well as against energetic insults in striatal neurons (10). Similarly,
creatine and in some cases the related analog cyclocreatine (CyCr)
exert protective effects in several animal models of neurodegenerative
diseases, like Huntington's disease (11), amyotrophic lateral
sclerosis (12), and a form of Parkinsonism (13). Likewise, Cr
pretreatment of cultured myotubes from dystrophic mdx mice
enhanced myotube formation and survival (14). Recent data indicate that
Cr reduces muscle necrosis and protects mitochondrial function in
vivo in mdx mice (15). Treatment of patients with Cr or
Cr analogs has, therefore, been proposed as a possible adjuvant therapy
for such diseases (16). The protection observed with Cr in these cell
and animal models may be explained partially by its function as a
cellular energy buffer and transport system via the PCr shuttle as
outlined above. An additional potential mechanism of Cr protection may
be linked to direct effects on mitochondrial permeability transition
(MPT) (17), which has been suggested to be a causative event in
different in vivo and in vitro models of cell
death (18-24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Oxygen consumption rates of liver mitochondria from transgenic
liver-mtCK mice in the presence of different creatine kinase substrates
Next, we measured, in transgenic mtCK-liver mitochondria, the opening
of the permeability transition pore induced by Ca2+ in the
presence of CK substrates, but in the complete absence of external
adenine nucleotides, and under different of conditions. To avoid any
effects caused by adenylate kinase activity, the specific adenylate
kinase inhibitor Ap5A was included in the medium in all
experiments. In the experiments shown in Fig.
1A, mitochondria were
energized with glutamate and malate in the absence of rotenone. Due to
the presence of 2 mM Mg2+ (but no EGTA) in the
medium, a rather high (120 µM) Ca2+ pulse had
to be administered to overcome MPT inhibition by magnesium. As shown in
Fig. 1A, Cr and CyCr effectively inhibited pore opening within the time frame of the experiment. In the absence of CK substrates or with GPA present, MPT pore opening occurred in a significant fraction of the mitochondrial population. These effects of
CK substrates were independent, at least qualitatively, on how the MPT
was triggered and of the respiratory substrates used as documented in
Fig. 1, B and C. In the experiments shown in Fig.
1B, mitochondria were again energized with complex I
substrates (glutamate and malate), but exposed to only 40 µM Ca2+ (which did not open the MPT per
se). Subsequent depolarization with 0.2 µM FCCP led
to rapid swelling of the mitochondria, both in the absence and presence
of CK substrates. Swelling was again sensitive to CsA, as well as to 50 µM ubiquinone 0, a novel and general MPT inhibitor (not
shown) (31), indicating opening of the MPT pore. Remarkably, however,
Cr and CyCr exerted MPT protection in a significant subpopulation of
mitochondria even in the absence of external adenine nucleotides and
under these very strongly pore-promoting conditions (absence of EGTA,
high phosphate, depolarization of mitochondria by FCCP, and presence of
complex I substrates). Again, GPA did not protect mitochondria from MPT
pore opening. Fig. 1C shows the same set of experiments but
with succinate-energized mitochondria in the presence of rotenone.
Here, the same qualitative conclusions can be drawn as from the data
presented in Fig. 1, A and B. In accordance with
the finding of Fontaine et al. (32), conditions are more
restrictive for MPT pore opening with complex II-linked substrates due
to decreased electron flux through complex I, a general property of the
MPT. With succinate as the electron donor, Cr and CyCr, but not GPA,
fully protected from MPT pore opening.
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Next we analyzed MPT protection by creatine at different calcium concentrations. In the experiments displayed in Fig. 1D, the Mg2+ concentration was reduced to 0.5 mM to compare better the effect of Cr as a function of the calcium load. Under these conditions, 30 µM Ca2+ did not open the MPT pore if Cr was present. Pore opening was, however, observed at higher Ca2+ concentrations (90 and 150 µM, solid curves in Fig. 1D). Nevertheless, Cr still inhibited significant fractions of mitochondria when compared with conditions without Cr but equal Ca2+ concentrations (dashed curves in Fig. 1D).
Based on these observations and the data presented in Table I, we
suggested that the protective effect on MPT pore opening seen with Cr
and CyCr as compared with GPA could be related to the kinetic
properties of these substrates, i.e. their rate of phosphorylation by mtCK. In contrast to GPA, Cr and CyCr are rapidly converted by mtCK via ATP to their respective phosphorylated compounds, PCr and CyPCr (30). If substrate phosphorylation by mtCK (with internally available ATP) were responsible for the observed effects, protection of MPT by Cr and CyCr should have been abolished under conditions where CK is inactive as an enzyme. As there is no absolutely specific CK inhibitor available, we omitted Mg2+ instead,
which is an essential cofactor for the CK reaction (33), from the
medium. With no Mg2+ present, creatine-stimulated
respiration with ATP was almost completely absent as shown in Fig.
2A (compare slopes at
arrow in trace a with that of trace
b). Importantly, in the absence of exogenous Mg2+,
state 3 respiration was still observed after addition of ADP (disodium
salt, Fig. 2A, trace a) indicating that
sufficient matrix Mg2+ is available for phosphorylation of
ADP by F0F1-ATP synthase, but this
Mg2+ is not accessible to mtCK located in the intermembrane
space. As shown in Fig. 2B, in the absence of
Mg2+, mitochondrial swelling induced by 20 µM
Ca2+ and 0.2 µM FCCP occurred to the same
extent regardless of whether CK substrates were present or not, in
contrast to what was observed in the presence of Mg2+ (see
the corresponding traces in Fig. 1B). MPT pore opening, however, was still fully blocked by 1 µM CsA, even in the
absence of Mg2+.
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In a further set of experiments, we analyzed the effect of
Mg2+ and Cr on MPT in more detail by varying the
concentrations of these substrates and measuring mtCK activity under
identical conditions. Representative swelling measurements at two
different Mg2+ concentrations with (solid
curves) and without (dashed curves) Cr are displayed in
Fig. 3A. From such curves we
determined the absorption difference (A540)
before and 4 min after triggering the MPT with 120 µM
Ca2+ (same conditions as in the experiments of Fig.
1A). The
A540 values were used to
calculate the fraction of swollen mitochondria with reference to the
A540 measured with 5 µM of the
channel-forming peptide alamethicin, which resulted in (not MPT-caused)
swelling of 100% of the mitochondria (not shown). As shown in Fig.
3C, the fraction of swollen mitochondria, i.e.
the fraction having undergone a permeability transition, decreased at
increasing Mg2+ concentrations, also in the absence of Cr
(open circles in Fig. 3C). This was to be
expected, as the MPT is regulated by an external inhibitory
Me2+-binding site (34). However, in the presence of 10 mM Cr, an additional protection starting at around 0.5 mM Mg2+ is observed (closed circles
in Fig. 3C). At this Mg2+ concentration, mtCK
activity, as measured in the pH-stat in the forward reaction with ATP
plus Cr, was at about 70% of the maximum value (open
triangles in Fig. 3C). Therefore, MPT protection by Cr
shows up at relatively high Mg2+ concentrations only (>0.5
mM) with corresponding high mtCK activities. A similar
analysis carried out by variation of the Cr concentration at constant
[Mg2+] (2 mM) revealed that MPT protection by
Cr is clearly a function of mtCK activity (Fig. 3, B and
D).
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These data are in strong support of the idea that the rate of
phosphorylation of CK substrates by active mtCK is related to their
effect on MPT. Phosphorylation of these CK substrates occurs in
microcompartments formed by mtCK and ANT (see below) (35). Because we
did not add external adenine nucleotides, phosphorylation must occur
via internally available ATP inside mitochondria, suggesting continuous
cycling of internal ADP and ATP between matrix and intermembrane space
mediated by ANT, if Cr or CyCr are present. As a consequence, if Cr is
present, we should expect a net production of PCr even in the absence
of exogenously added adenine nucleotides. This is indeed the case as
shown in Fig. 4A. Mitochondria
were incubated in the presence of 32Pi,
Mg2+, and 10 mM creatine. Only with energized
mitochondria and fully active mtCK, as well as with a working oxidative
phosphorylation system, we could observe a generation of PCr (Fig. 4,
lane a). These are exactly the conditions used in the
swelling experiments where MPT protection by Cr was observed (Fig. 1).
With de-energized mitochondria (Fig. 4, no substrate,
lane b), blocked ANT (20 µM atractyloside,
lane d), or blocked F0F1-ATP
synthase (1 µM oligomycin, lane e), no PCr was
produced. Note in lane c (absence of Mg2+), some
PCr is still generated due to residual Mg2+ (probably bound
to mtCK). Fig. 4B shows a time course experiment of net PCr
production by energized mitochondria in the presence of external Cr
plus Mg2+, but no adenine nucleotides. The measured adenine
nucleotide content is 0.11 ± 0.01 nmol per 25 µg of isolated
mitochondria from transgenic liver-mtCK mice. After a 40-min incubation
of succinate-energized mitochondria, 2.85 nmol of PCr were produced by
25 µg of mitochondria (Fig. 4B). Recalling that one ATP is consumed for every molecule of PCr produced, we can estimate that this
is over 25 times more than the measured adenine nucleotide content.
This quantitative consideration is a strong argument for internal
nucleotide cycling.
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Mitochondrial CK forms a microcompartment with ANT at the contact sites
(together with outer membrane porin), as well as along the cristae
(with ANT only (36, 37)). This compartmentation allows efficient
transphosphorylation of ATP to PCr and export of the latter to the
cytosol at peripheral contact sites. The reaction product ADP is fed
back via ANT to the matrix for rephosphorylation resulting in an
overall lowering of the apparent Km values of ADP
for oxidative phosphorylation (38). It is conceivable that
microcompartmentation of mtCK and ANT is also responsible for the
observed MPT inhibition by Cr and CyCr (48). To test this idea, we
measured MPT pore opening in liver mitochondria from control mice
lacking mtCK but with exogenously added recombinant human brain-type
dimeric CK (BB-CK). Under the conditions used, especially at pH 7.4, this cytosolic isoform does not bind to mitochondrial outer membranes
(Fig. 5A). The amount of BB-CK
enzyme activity added in these experiments was equivalent to that found (based on mtCK activity) within the mitochondria of transgenic liver-mtCK mice. Under these conditions, no protection of MPT pore
opening of control mitochondria with externally added CK by any of the
CK substrates was seen, even in the presence of Mg2+ (Fig.
5B, shown only for Cr). By contrast, with the same amount of
ubiquitous mtCK (the isoform expressed in the liver mitochondria of the
transgenic mice), significant binding of the enzyme to the surface of
mitochondria was observed (Fig. 5A). However, even with mtCK
bound to the outside of mitochondria, no noticeable MPT protection by
Cr was observed (Fig. 5C) as was also the case for BB-CK
(Fig. 5B). Even increasing the total amount of mtCK by
10-fold in the medium did not bring about detectable MPT protection by
Cr, although absolute binding of mtCK to the mitochondrial surface
increased by more than 3-fold under these conditions (Fig. 5A). Thus, addition of CK externally or even mtCK bound to
the outer membrane was not able to confer significant protection
against MPT.
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DISCUSSION |
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The present study was carried out to provide a mechanistic basis for the observation that certain substrates of creatine kinase efficiently inhibit the MPT (17). Because opening of the MPT pore appears to be causally related to cell death in several models (18-24), these studies may provide the basis for novel cytoprotective drugs. We have measured MPT pore opening in isolated mitochondria by the convenient swelling assay in sucrose-based medium and under different conditions, and we compared the response of transgenic mitochondria containing active or inactive CK with that of control mitochondria to which CK was added externally. These experiments revealed a major difference in MPT behavior. With liver mitochondria from transgenic mice, expressing mtCK in these organelles (39), but not with liver mitochondria from wild-type mice, we have observed MPT inhibition (based on measurements of uncoupled respiration) in the presence of Cr or CyCr in an earlier study (17). Here, by using mitochondrial swelling assays, we show that MPT protection by these CK substrates critically depends on two key factors.
The most striking new findings from the present study are that Cr protection of MPT requires magnesium, an essential cofactor for the CK reaction, and active mtCK at its proper location in the IMS. These observations together with the finding of net PCr production by isolated mitochondria, even in the absence of exogenous adenine nucleotides, suggest that endogenous adenine nucleotides are permanently cycling via ANT between matrix and IMS, if CK substrates are present that can efficiently be phosphorylated. This is the case for Cr and CyCr but not for GPA.
The consequences for MPT modulation by CK substrates could then be
explained by the influence of nucleotide binding to ANT and
conformational changes of this carrier (40, 41). To visualize this (see
Fig. 6), during adenine nucleotide
exchange, the common transport site for ADP and ATP on the ANT faces
alternatively the matrix (m) and cytosolic (c) side (42, 43).
Accordingly, the ANT changes its conformation between m- and c-state,
if conditions allow adenine nucleotide transport. In the absence of a
protonmotive force, the matrix ATP/ADP ratio of isolated mitochondria
is low (40, 44), and it is entirely conceivable that adenine
nucleotides are enzyme-bound (e.g. to the ATP synthase).
Certain (unknown) fractions of transport units (shown as ANT dimers in
Fig. 6, see also Ref 45) are locked either into the m- or c-state (Fig. 6A). Upon energization, the matrix ATP/ADP ratio rises, and
ATP will be liberated and transported to the IMS. There, ATP either gets diluted in the medium or is trapped by mtCK (Fig. 6B).
As a consequence, more transport units would now be in the c-state than
before energization. Under these conditions, MPT pore opening should be
favored, as seen with no CK substrate present. This situation is likely
to be similar to the effect of the strong ANT inhibitor, atractyloside,
known to stabilize the c-conformation and being an inducer of the MPT
(46). If, however, Cr (or CyCr) is present, the trapped ATP will be
transphosphorylated and the generated ADP transported to the matrix for
rephosphorylation (Fig. 6C). As this process proceeds, the
time-averaged fraction of transport units occupied with adenine
nucleotides and being in the m-state is expected to be higher, and
therefore, conditions for MPT pore opening are less favorable. In the
presence of GPA, the ATP delivered to the active site of mtCK is not
used up because GPA is not phosphorylated by mtCK (29). Consequently,
the ANT is largely locked into the c-conformation, again favoring MPT pore opening. At present, it is still unclear how ANT conformation would affect the MPT. An indirect effect, e.g. via the
surface potential, has been proposed (41, 47). Note the emphasis on microcompartmentation and functional coupling of mtCK and ANT as an
essential part of the model shown in Fig. 6. This is corroborated by
the fact that similar amounts of either cytosolic BB-CK or mtCK added
externally to the outside of control mitochondria, as is present as
mtCK in transgenic mitochondria, did not result in any significant Cr
protection of MPT.
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We have clearly demonstrated that in the presence of Cr, by measuring net production of PCr, oxidative phosphorylation proceeds even without adding external adenine nucleotides, although at a slow rate only. Nevertheless, besides the effect on the conformational state of the ANT, an additional contribution to MPT protection by Cr could be caused by variation of the matrix ATP/ADP ratio in favor of ADP (25), which is a strong MPT inhibitor (40, 44). On the other hand, the accumulating PCr is not believed to exert MPT inhibition (e.g. via the membrane potential) as we have shown earlier (17).
As mtCK functionally interacts with ANT at mitochondrial contact sites, as well as along the cristae membranes (36), a second possibility is that the two proteins may interact and that modulation by CK substrates may affect pore formation by the ANT. The known property of ANT to form an unspecific pore showing some of the characteristics of the MPT pore (48-50) in in vitro reconstituted systems has been taken by several authors as evidence that the ANT represents the central element of an MPT pore complex (51-59). However, it should be considered that pore formation is not unique to ANT but has also been described for other members of the mitochondrial carrier family (60, 61). Furthermore, mitochondria from ANT-deficient yeast still exhibit a mitochondrial multiconductance channel which is believed to be the electrophysiological counterpart of the MPT (62). A recent study by Linder et al. (63), showing that arginine modification has pronounced influences on MPT pore opening that are not modulated by the ANT ligands atractyloside and bongkrekic acid, further questions a direct involvement of the ANT in pore formation. Finally, the structural interactions between mtCK and ANT that were postulated in models of mitochondrial contact sites (64) still await experimental proof.
Creatine has been shown to exert strong protective effects against
glutamate and -amyloid toxicity and energetic insults in neuronal
cell cultures (9, 10), as well as in several animal models of
neurodegenerative diseases (11-13). It is interesting to note that in
the study of Brustovetsky et al. (10), Cr did not prevent
MPT in isolated brain mitochondria, despite protection from energetic
insults in cultured striatal neurons. This could be either due to the
low magnesium concentration (0.5 mM) used by these authors
in their assays or to the only weak coupling of the CK reaction to
oxidative phosphorylation in these particular preparations. For the
latter, however, no data were provided. In any case, both
interpretations are fully in line with the model concerning the effect
of Cr on MPT as proposed above. Thus, the beneficial effects of Cr seen
in animal disease models can be attributed to at least two mechanisms
that are ultimately linked by the PCr shuttle: 1) ATP levels and local
ATP/ADP ratios in the cytosol are kept high, thus sustaining membrane
ion gradients and other vital energy-consuming processes at a highly
efficient level, and 2) mitochondria are protected from MPT pore
opening via functional coupling of the mtCK reaction to oxidative phosphorylation.
Taken together, our findings offer important clues on MPT regulation by
mitochondrial ADP phosphorylation and may have interesting implications
for the design of creatine analogs to treat patients with
neurodegenerative diseases. In order to fully exploit the advantages of
the PCr shuttle for cell survival, such analogs would have to be
substrates for CK in both the forward and reverse direction of the reaction.
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ACKNOWLEDGEMENTS |
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We thank Elsa Zanolla for technical support and Dr. Alan P. Koretsky (National Institutes of Health, Bethesda) for providing transgenic liver-mtCK mice. We thank Dr. Alan P. Koretsky, Prof. Paolo Bernardi (University of Padua, Italy), and Dr. Laurence A. Kay (Université Joseph Fourier, Grenoble, France) for valuable discussions and comments on the manuscript.
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FOOTNOTES |
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* This work was supported by private sponsoring (Careal Holding AG, Zürich, Switzerland, and AVICENA Inc., Boston (Dr. Rima Kaddurah-Daouk)) (to M. D. and T. W.), a grant from the Swiss Society for Research on Muscle Diseases (to B. W., O. S., and T. W.), and by Swiss National Science Foundation Grant 31-62024.00 (to T. W. and U. S.).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.
To whom correspondence should be addressed: Institute of
Biotechnology, Swiss Federal Institute of Technology,
ETH-Hönggerberg, CH-8093 Zürich, Switzerland. Tel.:
41-1-633-36-36; Fax: 41-1-633-10-51; E-mail:
dolder@biotech.biol.ethz.ch.
§ Present address: Coty-Lancaster Group, International Research and Development Center, Athos Palace/2, Rue de la Lùjerneta, MC-98000 Monaco.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M208705200
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ABBREVIATIONS |
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The abbreviations used are:
CK, creatine kinase;
ANT, adenine nucleotide translocase;
Ap5A, P1,P5-di(adenosine
5')-pentaphosphate (an inhibitor of adenylate kinase);
BB-CK, cytosolic
brain-type creatine kinase;
Cr, creatine;
CsA, cyclosporin A;
CyCr, cyclocreatine;
FCCP, carbonyl cyanide
p-trifluoromethoxyphenylhydrazone;
GPA, -guanidinopropionic acid;
IMS, intermembrane space;
MPT, mitochondrial permeability transition;
mtCK, mitochondrial creatine
kinase;
PCr, phosphocreatine;
Mops, 4-morpholinepropanesulfonic
acid.
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REFERENCES |
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