Here, we present a multiparameter study on the effects of MC on isolated rat liver mitochondria RLM suspended either in a KCl-based or in a sucrose-based medium.. In KCl-based medium, bu
Trang 1Kathleen Kupsch1,2, Silvia Hertel3, Peter Kreutzmann1,2, Gerald Wolf2, Claus-Werner Wallesch3, Detlef Siemen3,* and Peter Scho¨nfeld1,*
1 Institute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Germany
2 Institute of Medical Neurobiology, Otto-von-Guericke University Magdeburg, Germany
3 Department of Neurology, Otto-von-Guericke University Magdeburg, Germany
Minocycline (MC) belongs to the tetracycline (TC)
family of antibiotics, which block the protein synthesis
of the bacterial ribosome [1] It is commonly used in
the treatment of diseases with an inflammatory
back-ground [2], but MC possesses cytoprotective properties
as well Thus, treatment with MC has been shown to
be beneficial in animal models of numerous
neuro-degenerative diseases [3–6] and of cerebral and cardiac
ischemia [7,8] However, the efficacy of its
neuro-protective actions remains controversial [9] The cyto-protective properties of MC are discussed in terms of anti-inflammatory [10], antioxidative [11] and antiapop-totic activities [4,8,12,13], but MC is also considered to
be an inhibitor of the poly-(ADP-ribose) polymerase-1 [14] and of matrix metalloproteinases [15] The antia-poptotic effect of MC has been attributed to upregula-tion of the antiapoptotic protein Bcl-2 [12,13], reduced expression of caspases [8,16], and inhibition of the
Keywords
magnesium; minocycline; mitochondria;
neuroprotection; permeability transition
Correspondence
K Kupsch, Institute of Biochemistry and
Cell Biology, Otto-von-Guericke University
Magdeburg, Leipziger Str 44, D-39120
Magdeburg, Germany
Fax: +49 391 6714365
Tel: +49 391 6714362
E-mail: kkupsch@web.de
*These authors contributed equally to this
work
(Received 7 November 2008, revised
8 January 2009, accepted 13 January 2009)
doi:10.1111/j.1742-4658.2009.06904.x
There is an ongoing debate on the presence of beneficial effects of mino-cycline (MC), a tetramino-cycline-like antibiotic, on the preservation of mito-chondrial functions under conditions promoting mitochondria-mediated apoptosis Here, we present a multiparameter study on the effects of MC
on isolated rat liver mitochondria (RLM) suspended either in a KCl-based
or in a sucrose-based medium We found that the incubation medium used strongly affects the response of RLM to MC In KCl-based medium, but not in sucrose-based medium, MC triggered mitochondrial swelling and cytochrome c release MC-dependent swelling was associated with mito-chondrial depolarization and a decrease in state 3 as well as uncoupled respiration Swelling of RLM in KCl-based medium indicates that MC per-meabilizes the inner mitochondrial membrane (IMM) to K+and Cl) This view is supported by our findings that MC-induced swelling in the KCl-based medium was partly suppressed by N,N¢-dicyclohexylcarbodiimide (an inhibitor of IMM-linked K+-transport) and tributyltin (an inhibitor of the inner membrane anion channel) and that swelling was less pronounced when RLM were suspended in choline chloride-based medium In addition,
we observed a rapid MC-induced depletion of endogenous Mg2+ from RLM, an event that is known to activate ion-conducting pathways within the IMM Moreover, MC abolished the Ca2+ retention capacity of RLM irrespective of the incubation medium used, most likely by triggering per-meability transition In summary, we found that MC at low micromolar concentrations impairs several energy-dependent functions of mitochondria
in vitro
Abbreviations
CaG, Calcium Green-5N; CholCl, choline chloride; CRC, Ca 2+ retention capacity; CsA, cyclosporin A; IMAC, inner membrane anion channel; IMM, inner mitochondrial membrane; MC, minocycline; MgG, Magnesium Green; mPTP, mitochondrial permeability transition pore; RLM, rat liver mitochondria; SEM, standard error of the mean; TBT, tributyltin chloride; TC, tetracycline; Dwm, mitochondrial membrane potential.
Trang 2release of proapoptotic proteins from mitochondria
[8,17] This latter activity of MC might result from its
ability to block opening of the mitochondrial
perme-ability transition pore (mPTP [3,4,18]), a
large-conduc-tance megachannel in the inner mitochondrial
membrane (IMM) [19]
Recent studies, however, challenge the view that MC
is an inhibitor of the mPTP, and instead report
detri-mental effects of MC on mitochondrial physiology
[20–22] Hence, there is ongoing debate as to whether
or not suppression of mPTP opening is involved in
MC-related cytoprotection In order to contribute to a
better understanding of mitochondria-targeted actions
of MC, we investigated the effect of MC on various
energy-related parameters of isolated rat liver
mito-chondria (RLM) As the controversial data reported to
date were obtained using either ionic or nonionic
incu-bation media, we focused our attention on the role of
the composition of the incubation medium in
MC-linked activities We observed that MC exerted
several detrimental effects on mitochondrial properties
such as respiration and mitochondrial membrane
potential (Dwm) when RLM were incubated in a
KCl-based medium In contrast, these parameters were not
affected by MC when RLM were incubated in a
sucrose-based medium However, irrespective of the
incubation medium used, MC decreased the
mitochon-drial Ca2+ retention capacity (CRC) and, in addition,
induced a leakage of matrix Mg2+ We propose that
mitochondria are primarily affected by two activities
of MC: (a) depletion of endogenous Mg2+; and (b)
opening of the mPTP in Ca2+-loaded RLM
Results
Swelling behavior
A first hint that the surrounding medium influences
the response of RLM to MC came from the
observa-tion that MC induced a swelling response that differed
with the incubation medium used (Fig 1A) Thus,
RLM suspended in the sucrose-based medium did not
swell upon treatment with MC, even at concentrations
up to 100 lm In contrast, MC at concentrations
‡ 25 lm triggered a rapid decrease in light absorbance
in the KCl-based medium, indicating expansion of the
mitochondrial matrix volume The extent of swelling
was concentration-dependent and was not affected by
the potent mPTP inhibitor cyclosporin A (CsA, not
shown) Swelling was paralleled by a release of
cyto-chrome c from RLM into the medium, which also
could not be blocked by CsA (Fig 1B) In the
sucrose-based medium, MC did not induce the translocation of
cytochrome c (Fig 1C) In contrast, cytochrome c release was similar in both media when the trans-location was triggered by activating the mPTP using calcium ions
Membrane potential and mitochondrial respiration
The MC-induced swelling of RLM in KCl-based med-ium was associated with depolarization of the IMM (Fig 2A) When RLM were suspended in medium supplemented with safranine O as a Dwm probe, they rapidly accumulated the cationic dye, as indicated by the dramatic decrease of the safranine O fluorescence upon addition of RLM (Fig 2A) In the KCl-based medium, MC in concentrations found to induce
A
B
C
Fig 1 Effect of MC on swelling behavior and cytochrome c release RLM (0.5 mgÆmL)1protein) were suspended in either KCl-based (black traces) or sucrose-KCl-based (gray trace) medium (A) RLM were treated with MC as indicated Data shown are mean ± stan-dard error of the mean (SEM) of three independent preparations (B, C) After treatment of RLM (0.5 mgÆmL)1protein) incubated in KCl-based (B) or sucrose-based (C) medium with 100 l M MC or
200 l M CaCl 2 , cytochrome c was measured in the mitochondrial pellet and the supernatant as described in Experimental procedures Representative blots of three preparations are shown.
Trang 3swelling triggered a strong increase in fluorescence,
reflecting the release of safranine O from the
mito-chondria due to a decreased Dwm In contrast, when
RLM were incubated in the sucrose-based medium,
MC-induced depolarization of the IMM was minor,
even at relatively high concentrations of MC (100 lm)
Furthermore, MC affected the respiration of RLM
when they were suspended in KCl-based medium, but
not in sucrose-based medium In the absence of MC,
the rates of state 3 respiration were 56 ± 7 (1 mm
Mg2+) and 62 ± 6 (Mg2+-free) nmol O2Æmin)1Æmg)1 protein in KCl-based medium, or 51 ± 6 (1 mm
Mg2+) and 49 ± 3 (Mg2+-free) nmol O2Æmin)1Æmg)1 protein in sucrose-based medium As shown in Fig 2B, RLM exposed to MC (25–100 lm) exhibited a concentration-dependent decrease of state 3 respira-tion At the highest concentration applied (100 lm),
MC decreased state 3 respiration to 60% of the con-trol (without MC) The decline of state 3 respiration was dependent on the Mg2+concentration in the med-ium, with MC being more effective in the absence of
Mg2+ In Mg2+-free medium, state 3 respiration decreased to about 40% of the control upon treatment with 100 lm MC Mg2+ did not affect state 3 respira-tion in the absence of MC MC also inhibited the carbonyl cyanide p-(trifluoromethoxy)-phenylhydraz-one-uncoupled respiration of RLM in KCl-based medium (not shown) In contrast, state 3 respiration of RLM suspended in sucrose-based medium was not affected by MC (Fig 2C) The decline of state 3 and carbonyl cyanide p-(trifluoromethoxy)-phenylhydraz-one-dependent respiration was found not only when mitochondria oxidized NAD-dependent substrates (glutamate and malate), but also with succinate (plus rotenone) Thus, 100 lm MC decreased succinate-supported state 3 respiration from 95 ± 7 nmol
O2Æmin)1Æmg)1 protein to 68 ± 5 nmol O2Æmin)1Æmg)1 protein
CRC
As recent results concerning the role of MC in Ca2+ -triggered permeability transition were controversial [20–22], we studied the effect of MC on the mitochon-drial CRC, which indicates the susceptibility of mito-chondria to undergo permeability transition upon
Ca2+ uptake into the mitochondrial matrix Low concentrations of MC (10 lm) completely abolished the ability of RLM to accumulate Ca2+from the KCl-based medium (Fig 3A) The inability of MC-treated RLM to accumulate Ca2+ in the KCl-based medium can simply be explained by the MC-induced swelling and concomitant decrease of Dwm, the driving force for Ca2+uptake
However, Ca2+ uptake was also suppressed by MC
in the sucrose-based medium, where MC did not initi-ate swelling or a collapse of Dwm Under these condi-tions, slightly higher concentrations of MC were needed to completely abolish Ca2+ uptake (‡ 50 lm; Fig 3B) How can we explain the MC-induced reduc-tion in CRC in the absence of mitochondrial depolar-ization? In order to clarify this issue, RLM were incubated in sucrose-based medium supplemented with
A
B
C
Fig 2 Effect of MC on the membrane potential and respiration.
(A) In order to follow changes in Dw m , RLM (0.5 mgÆmL)1protein)
were suspended in either KCl-based (black traces) or sucrose-based
(gray trace) medium supplemented with 5 l M safranine O as Dwm
probe After the uptake of safranine O by energized RLM, MC was
added as indicated Representative traces of a single preparation
out of three preparations are shown (B, C) RLM (1 mgÆmL)1
protein) suspended in either KCl-based (B) or sucrose-based (C)
medium were pretreated for 2 min with 25, 50, 75 or 100 l M MC.
State 3 respiration was stimulated by the addition of 2 m M ADP.
Respiration was also measured in the absence of Mg 2+ Data
shown (mean ± SEM) were obtained from three to four
pre-parations.
Trang 4CsA to prevent Ca2+-triggered mPTP opening
(Fig 4A, ‘control’ trace) We found that addition of
MC (25 lm) to RLM preloaded with Ca2+ (100
nmo-lÆmg)1 protein) initiated the release of Ca2+
MC-induced Ca2+release was paralleled by depolarization
of the IMM (Fig 4B)
MC initiated the oxidation of external NADH
We were now interested to understand why MC
decreased the state 3 respiration of RLM in KCl-based
medium (Fig 2B) As uncoupled respiration was also
sensitive to MC (not shown), we can exclude the
possi-bility that MC decreased state 3 respiration by
block-ing the F1F0-ATPase or ADP⁄ ATP exchange across
the IMM The observed loss of cytochrome c from
RLM upon treatment with MC, however, could
con-tribute to the MC-induced decline in respiration
Therefore, we examined the effect of added
cyto-chrome c on the respiration of MC-treated RLM
Addition of cytochrome c (5 lm) increased the
respira-tion only moderately (Fig 5A) Surprisingly, subse-quent addition of 200 lm NADH (substrate of complex I) strongly increased the respiration of MC-treated RLM, which was not sensitive to CsA (not shown) In the absence of MC, addition of cyto-chrome c and NADH only slightly affected mitochon-drial respiration (Fig 5B) However, subsequent addition of MC dramatically stimulated O2 consump-tion Stimulation of respiration by external NADH suggests that MC permeabilized the IMM to NADH; the mechanism of this remains unclear
MC depleted mitochondria of endogenous Mg2+
MC is a highly lipophilic TC derivative, and it is worth recalling that TCs are able to chelate polycharged
A
B
Fig 3 Effect of MC on CRC RLM (0.5 mgÆmL)1 protein) were
suspended in either KCl-based (A) or sucrose-based (B) medium
supplemented with 200 l M ADP and 1 lgÆmL)1of the F 1 F 0 -ATPase
inhibitor oligomycin Aliquots (5 lL) of a 5 m M CaCl2solution were
added The extramitochondrial Ca 2+ concentration was measured
with CaG as Ca 2+ fluorochrome Representative traces of a single
preparation out of three preparations are shown.
A
B
Fig 4 Effect of MC on CRC and Dwmof Ca 2+ -loaded mitochondria RLM (0.5 mg of protein) were suspended in sucrose-based med-ium supplemented with 1 l M CsA (A) MC at 25 l M was added to RLM loaded with 100 nmol Ca2+⁄ mg protein (indicated by two
25 l M Ca 2+ additions; solid line) The increase of the Ca 2+ –CaG flu-orescence observed after addition of MC indicates Ca 2+ release from RLM Ca2+uptake by RLM in the absence of MC (solid and dotted lines) is shown for comparison (B) The traces show the cor-responding responses of Dwm to the addition of Ca 2+ and ⁄ or MC Representative traces of a single preparation out of three prepara-tions are shown.
Trang 5cations, including Mg2+ [23,24] Therefore, there is
reason to assume that MC could extract Mg2+ from
RLM In order to investigate this, we tested the effect
of MC on the matrix Mg2+ content using the Mg2+
-specific dye Magnesium Green (MgG) Figure 6A
shows that addition of MC decreased the fluorescence
of the matrix Mg2+–MgG complex in a
concentration-dependent manner (Fig 6A) This fluorescence
decrease suggests that MC has the capability to deplete
RLM of Mg2+ This view is supported by the finding
that the bivalent cation ionophore A23187 induced a
similar decrease in Mg2+–MgG fluorescence It should
be noted that MC also decreased the fluorescence of
the Mg2+–MgG complex when RLM were suspended
in the sucrose-based medium (Fig 6B) In addition, we observed that TC induced a much smaller decrease in
Mg2+–MgG fluorescence than did MC (Fig 6B)
Inhibition of MC-induced swelling MC-induced Mg2+ depletion of RLM was paralleled
by mitochondrial swelling in KCl-based medium, but not in sucrose-based medium (Fig 1A) What could be the mechanism underlying MC-triggered swelling of RLM in the KCl-based medium? Mg2+ depletion is known to activate ion-conducting pathways within the IMM, such as the inner membrane anion channel (IMAC), the K+-uniporter, and the K+⁄ H+
-antiport-er [25–27] Th-antiport-erefore, we studied a possible effect of inhibitors of these ion-conducting pathways Indeed, N,N¢-dicyclohexylcarbodiimide (1 lm), a nonspecific inhibitor of both the mitochondrial K+-uniporter [28] and the mitochondrial K+⁄ H+-antiporter [29],
moder-Fig 5 Effect of cytochrome c and NADH on mitochondrial
respira-tion in the presence and absence of MC RLM (1 mgÆmL)1protein)
were suspended in KCl-based medium Traces of the oxygen
con-centration in the medium (trace a) and its first derivative (d[O2] ⁄ dt;
trace b) are shown (A, B) The respiration of control and MC-treated
(100 l M ) RLM in response to additions of 5 l M cytochrome c
(Cyt c) and 100 l M NADH is shown Rates of respiration are given
as numbers (in nmol O2Æmin)1Æmg)1protein) at the d[O2] ⁄ dt traces.
Representative experiments obtained from four mitochondrial
prep-arations are shown.
A
B
Fig 6 Effect of MC on mitochondrial Mg2+ content RLM (1 mgÆmL)1 protein) loaded with MgG were suspended either in KCl-based or sucrose-based medium supplemented with 1 m M EDTA MC was added at the indicated concentrations (A) The decrease of the Mg 2+ –MgG fluorescence indicates release of endogenous Mg 2+ from RLM incubated in KCl-based medium A23187 (1 l M ) was applied to induce complete depletion of matrix
Mg2+ Representative traces obtained from four mitochondrial prep-arations are shown (B) The graph summarizes the MC-induced
Mg 2+ release from RLM in KCl-based and sucrose-based medium.
Mg2+ depletion triggered by TC is also included for comparison (n = 3) Data are mean ± SEM of four preparations (*P < 0.05,
**P < 0.01, ***P < 0.001).
Trang 6ately reduced the MC-induced swelling of RLM
(Fig 7A,D) Similarly, treatment of RLM with 1 lm
of the IMAC inhibitor tributyltin chloride (TBT) [30]
inhibited MC-induced swelling (Fig 7B,D) Finally,
when RLM were suspended in choline chloride
(Chol-Cl)-based medium, only minor swelling was observed
upon addition of 100 lm MC (Fig 7C,D) This
obser-vation might indicate that the choline cation is a poor
substrate of the K+-uniporter [31]
Discussion
We have demonstrated here that MC impairs
mito-chondrial energy metabolism Our results support
recent reports proposing that MC most likely has no
beneficial effects on mitochondria [21,32]
Further-more, we show that the response of energy-linked
parameters (state 3 respiration, Dwm) to MC depends
on the mitochondrial environment When RLM were
suspended in KCl-based medium, MC triggered
swell-ing and decreased state 3 respiration as well as Dwm A
similar observation has been reported after treatment
of rat brain mitochondria with MC in KCl-based med-ium [21,22] In sucrose-based medmed-ium, however, we did not observe any effect of MC on swelling behavior, state 3 respiration or Dwm of RLM Conflicting with our results, MC-induced swelling of RLM suspended
in mannitol⁄ sucrose-based medium has been reported elsewhere [32] The reason for this discrepancy remains unclear
What could be the mechanism underlying the MC-induced decline of state 3 respiration, breakdown of
Dwm and swelling of RLM suspended in KCl-based medium? There is reason to assume that these changes are associated with depletion of mitochondrial matrix
Mg2+ Depletion of Mg2+ from RLM could be explained by the high lipophilicity of MC (chloro-form⁄ water partition coefficient of 30 at pH 7.4 [24]) and the ability of MC to chelate bivalent cations such
as Mg2+[23] Mg2+depletion is known to activate the IMAC, the mitochondrial K+-uniporter, and the mito-chondrial K+⁄ H+-antiporter, ion-conducting path-ways that are normally blocked in vitro by Mg2+ binding [25–27,33] These well-known observations
Fig 7 Effect of N,N¢-dicyclohexylcarbodiimide, TBT and CholCl on MC-induced swelling RLM (0.5 mgÆmL)1protein) were suspended in KCl-based or CholCl-based medium Data shown are mean ± SEM (n = 3) (A) MC at 100 l M was added to RLM pretreated in KCl-based medium with N,N¢-dicyclohexylcarbodiimide (an inhibitor of the K + -uniporter and the K + ⁄ H + -antiporter) for 10 min (B) The suppression of the MC-induced swelling by 1 l M TBT (an inhibitor of the IMAC) is shown (C) MC was added to RLM suspended in CholCl-based medium (D) Statistical analysis of the absorbance values 2 min after addition of MC (dotted line in A–C): (A) the MC-induced absorbance decrease was significantly smaller in CholCl-based medium (82.4 ± 2.6%) than in KCl-based medium (65.1 ± 3.8% of baseline; P < 0.05, n = 4); (B, C) Both TBT and N,N¢-dicyclohexylcarbodiimide significantly restored the absorbance of MC-treated RLM (for TBT, 87.4 ± 0.7 versus 75.6 ± 2.7, P < 0.01, n = 4; for N,N¢-dicyclohexylcarbodiimide, 82.8 ± 1.9 versus 75.1 ± 2.6, P < 0.05, n = 4).
Trang 7inspired us to assume that MC unmasks these
ion-con-ducting pathways, thereby enabling the uptake of KCl
by RLM This hypothesis is in line with our finding
that MC depletes mitochondria of Mg2+in both ionic
and nonionic media equally, whereas MC-induced
swelling occurred only in KCl-based medium
Addi-tionally, we found that TC, which has a much smaller
effect on matrix Mg2+ concentration than does MC,
does not trigger swelling (not shown) Furthermore, it
is known that bivalent cations react with TCs to form
fluorescent chelates [34], which are mainly found in the
mitochondrial and in the microsomal fractions [35]
TCs preferentially bind to cations on membrane
sur-faces [34] It is also worth mentioning that other
reagents, such as mercurials
(p-hydroxymercuribenzo-ate) and nonesterified long chain fatty acids, deplete
RLM of endogenous Mg2+as well, and hence induce
large-amplitude swelling in KCl-based medium
[31,36,37]
In addition to the observed partial loss of the
elec-tron carrier cytochrome c from RLM, limitation of
NADH oxidation could contribute to the decline in
state 3 respiration Such a possibility is suggested
from our finding that the basal respiration of
MC-treated RLM strongly responds to external NADH
Keeping in mind that the IMM of intact RLM is
impermeable to NADH, this surprising observation
could indicate that MC induced leakage of NADH
from the matrix
There are controversial reports on whether or not
MC protects mitochondria against Ca2+-triggered
opening of the mPTP It is known that MC fails to
protect mitochondria against toxin-stimulated
perme-ability transition [32] It has also been shown that MC
cannot prevent Ca2+-triggered swelling when energized
RBM are in sucrose-based medium [21] Similarly,
cytochrome c release initiated by Ca2+-triggered
per-meability transition was not prevented by MC [21] In
contrast, other studies conclude that MC prevents the
Ca2+-dependent permeability transition irrespective of
the medium used [20,22] This conclusion was derived
from the observation that MC suppressed Ca2+
-depen-dent swelling However, suppression of swelling was
associated with deficient Ca2+ uptake [20,22] and a
collapse of Dwm [22] Therefore, the suppression of
swelling might be due to the lower sensitivity of
de-energized mitochondria to undergo Ca2+-triggered
permeability transition Here, we confirm that MC
abolishes the Ca2+uptake of RLM suspended in
KCl-based or sucrose-KCl-based medium We also demonstrate
that RLM preloaded with Ca2+ release the
accumu-lated Ca2+upon addition of MC, even in the presence
of CsA The release of Ca2+ is paralleled by
depolar-ization Taken together, these observations suggest that mitochondrial Ca2+uptake is not primarily inhib-ited by MC Instead, MC seems to trigger a CsA-insensitive permeability transition in Ca2+-loaded RLM incubated in sucrose-based medium This effect might be explained by the ability of MC to deplete mitochondria of endogenous Mg2+, as Mg2+ is a powerful inhibitor of mPTP formation [38]
Our data suggest that prior results concerning the action of MC on the mPTP might have been misinter-preted Thus, we show that the previously reported MC-related inhibition of Ca2+-triggered swelling in sucrose-based medium does not reflect inhibition of the mPTP Furthermore, our results demonstrate that the effects of a drug on mitochondrial parameters can depend on the incubation medium used For instance, the operation of K+-dependent ion-conducting pathways embedded in the IMM is excluded when sucrose-based medium is applied Hence, the medium composition should be more carefully considered in future studies In general, a KCl-based medium mimics the in vivo situation much better than a sucrose-based medium
In summary, we propose that MC impairs the function of isolated mitochondria by two distinct mechanisms: (a) it depletes mitochondria of endoge-nous Mg2+, thereby inducing permeability of the IMM to K+ and Cl); and (b) it activates the mPTP
in the presence of external Ca2+ or in Ca2+-loaded RLM, thereby inducing permeability of the IMM to nonionic, low-molecular solutes, such as sucrose These detrimental activities might contribute to the harmful effects of MC, as recently reported from a phase III clinical trial in patients with amyotrophic lateral sclerosis [39] In the study cited, doses of
400 mgÆday)1 were administered Assuming a body weight of 70 kg and a body water content of 60%, the concentration of MC in the aqueous phase can
be calculated to be about 20 lm at most after a bolus administration Considering that MC easily permeates the blood–brain barrier [9] and has high solubility in membranes [24], there is good reason to assume that mitochondria can be affected by harmful activities of MC in vivo
Experimental procedures
Chemicals CsA was obtained from Alexis (Lausanne, Switzerland) CaG and MgG were from Molecular Probes (Karlsruhe, Germany) All other biochemicals were purchased from Sigma (Steinheim, Germany)
Trang 8Procedures for animal use were in strict accordance with
the Animal Health and Care Committee of the State
Sach-sen-Anhalt, Germany Male Wistar rats
(Harlan–Winkel-mann, Borchen, Germany) were single-housed and
maintained under a 12 : 12 h light⁄ dark cycle Before being
killed, rats were allowed a 2 week acclimation period and
had free access to standard food and water ad libitum
Isolation of RLM
RLM were prepared from Harlan-Winkelmann male Wistar
rats (Borchen, Germany) with a wet-liver mass of about 8 g
as described previously [40] The final mitochondrial pellet
was suspended in isolation medium (250 mm sucrose,
0.5 mm EDTA; pH 7.4) Mitochondrial protein in the stock
suspension was determined using the Biuret method The
total yield of isolated mitochondria per liver was about
120 mg of protein The respiratory control ratio was
routinely measured to be in the range 6–12
Incubations
Experiments were performed in two different incubation
media, a KCl-based (125 mm KCl, 20 mm Tris, 1 mm
MgCl2, 10 lm EGTA, 5 mm glutamate, 5 mm malate, and
1 mm Pi; pH 7.2) or a sucrose-based (200 mm sucrose,
10 mm Tris, 1 mm MgCl2, 10 lm EGTA, 5 mm glutamate,
5 mm malate, and 1 mm Pi; pH 7.2) medium Further
addi-tions are specified in the figure legends MC was added
from an aqueous stock solution (10 mm) Incubations were
routinely performed at 30C
Measurement of swelling and cytochrome c
release
Swelling of mitochondria was measured as decrease in light
absorbance at 620 nm using a multiplate reader (Titertek
Plus MS212; ICN, Frankfurt, Germany) RLM were
sus-pended in 200 lL of the indicated incubation medium
(0.5 mgÆmL)1 protein) For determination of cytochrome c
release, RLM (0.5 mgÆmL)1 protein) were preincubated in
1 mL of the incubation medium for 5 min Subsequently,
MC or Ca2+ was added as indicated, and mitochondria
were incubated at room temperature for an additional
5 min After centrifugation of the incubation mixture
(4000 g, 5 min), the supernatants (extramitochondrial
frac-tions) were collected The pellets (mitochondrial fracfrac-tions)
were resuspended in 250 lL of 10% SDS and incubated at
95C for 10 min All fractions were diluted 1 : 4 in
Roti-Load 4x and denatured at 95C for 5 min Equal volumes
were applied to a 5–20% SDS gel After electrophoresis
(20 mAÆgel)1, 90 min), proteins were transferred to a
Hybond-c Extra nitrocellulose membrane (Amersham Bio-sciences, Little Chalfont, UK) Immunostaining was per-formed using the primary 7H8.2C12 mouse antibody against cytochrome c (1 : 500; BD Pharmingen) plus secondary goat anti-mouse IgG + IgM conjugated to peroxidase (1 : 10 000; Jackson ImmunoResearch Laboratories Inc., Westgrove, USA) Protein bands were visualized by chemilu-minescence (Immobilon Western, Millipore, Billerica, USA)
Membrane potential and CRC Membrane potential (Dwm) and extramitochondrial Ca2+ concentration were recorded using safranine O (Dwm probe) and the membrane-impermeant Ca2+-sensitive dye Calcium Green-5N (CaG) RLM (0.5 mg of protein) were suspended in 1 mL of the indicated incubation medium supplemented with 5 lm safranine O or 100 nm CaG Flu-orescence intensities were measured at excitation wave-lengths of 525 and 506 nm and emission wavewave-lengths of
587 and 532 nm for safranine O and CaG, respectively, using a Cary Eclipse fluorometer (Varian, Darmstadt, Germany)
Respiration Oxygen consumption was measured in 2 mL of incubation medium (1 mgÆmL)1 protein) at 30C using the high-reso-lution OROBOROS Oxygraph (Anton Paar KG, Graz, Austria) State 3 respiration and uncoupled respiration was adjusted by addition of 2 mm ADP and 0.7 lm p-(trifluoro-methoxy)-phenylhydrazone, respectively
Determination of matrix Mg2+
Free matrix Mg2+ was monitored fluorimetrically using MgG as described previously [36] Briefly, mitochondria suspended in isolation medium (10 mgÆmL)1) were loaded with MgG-acetoxymethylester (2 lm) for 5 min at room temperature After centrifugation of the mitochondrial sus-pension (10 000 g for 2 min), the mitochondrial pellet was resuspended in 450 lL of the isolation medium Aliquots of MgG-loaded RLM (0.2 mg of protein) were added to 1 mL
of the indicated assay medium supplemented with 1 mm EDTA The fluorescence was recorded using a PerkinElmer Luminescence Spectrophotometer LS50B at 510 nm excita-tion and 535 nm emission
Statistical analysis All experiments were replicated in at least three indepen-dent mitochondrial preparations Values obtained were compared by one-way ANOVA followed by Dunnett post-test using graphpadprism (version 3.02; GraphPad Software, San Diego, CA, USA)
Trang 9This work was supported by funding from
Magde-burger Forschungsverbund NBL3 (to G Wolf and
D Siemen) and from the BMBF (to D Siemen)
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