(Received for publication, November 27, 1995; and in revised form, January 12, 1996)
From the
After accumulation of a Ca load, the addition
of uncoupler to respiring rat liver mitochondria is followed by opening
of the permeability transition pore (MTP), a voltage-dependent channel
sensitive to cyclosporin A. The channel's voltage threshold is
profoundly affected under conditions of oxidative stress, with a shift
to more negative values that may cause MTP opening at physiological
membrane potentials. In this paper we further clarify the mechanisms by
which oxidative agents affect the apparent voltage dependence of the
MTP. We show that two sites can be experimentally distinguished. (i) A
first site is in apparent oxidation-reduction equilibrium with the
pyridine nucleotide (PN) pool (NADH/NAD + NADPH/NADP); PN
oxidation is matched by increased MTP open probability under conditions
where the glutathione pool is kept in the fully reduced state; this
site can be blocked by N-ethylmaleimide but not by
monobromobimane, a thiol-selective reagent. (ii) A second site
coincides with the oxidation-reduction-sensitive dithiol we have
recently identified (Petronilli, V., Costantini, P., Scorrano, L.,
Colonna, R., Passamonti, S., and Bernardi, P.(1994) J. Biol. Chem. 269, 16638-16642); dithiol cross-linking at this site by
arsenite or phenylarsine oxide is matched by increased MTP open
probability under conditions where the PN pool is kept in the fully
reduced state; at variance from the first, this site can be blocked by
both N-ethylmaleimide and monobromobimane and is probably in
equilibrium with the glutathione pool. Based on these findings, we
reassess the mechanisms by which many oxidative agents affect the MTP
and resolve conflicting reports on the relative role of PN and
glutathione oxidation in the permeability transition within the
framework of MTP (dys)regulation at two separate sites.
Mitochondrial function is affected by oxidative stress, as shown
by the permeability increase caused by a wide variety of oxidants ( (1) and references therein). The permeability increase is
favored by Ca accumulation and causes equilibration
of solutes up to
1500 Da in molecular mass, depolarization with
uncoupling, and release of the previously accumulated Ca
(see (2, 3, 4) for recent reviews).
Most authors now agree that this phenomenon (the ``permeability
transition'' of Hunter and Haworth (5, 6, 7) ) is due to opening of the
permeability transition pore (MTP). (
)The MTP is an inner
membrane channel (5, 6, 7) inhibited by
CsA(8, 9, 10, 11) , which, by a
number of criteria (12, 13, 14, 15) , appears to
coincide with the mitochondrial megachannel discovered by patch clamp
studies of rat liver mitoplasts(16, 17, 18) .
The mechanism(s) by which oxidants induce opening of the MTP has
been the subject of many studies, often carried out without realizing
that the permeability transition was at least partially involved (see
the thorough discussion by Zoratti and Szabo'(4)). Work by
Lehninger and co-workers pointed to the role of the oxidation-reduction
level of PN in the modulation of mitochondrial Ca fluxes, showing that PN oxidation caused Ca
efflux, which could be reversed by PN reduction(19) .
Several subsequent studies, however, showed that Ca
efflux and the redox state of PN could be dissociated (20, 21) and that PN oxidation could be the
consequence rather than the cause of the permeability transition and
therefore of Ca
efflux(22) . These
observations undermined the PN hypothesis of regulation of
Ca
efflux, now ascribed to MTP opening(4) .
Another potential target for oxidant species that received
considerable attention in MTP (dys)regulation is mitochondrial
glutathione ( (4) and references therein). In an influential
study, Pfeiffer and co-workers (23) were able to independently
modulate the NAD/NADH, NADP/NADPH, and GSSG/GSH ratios and concluded
that the latter was the relevant factor since permeabilization could be
observed after glutathione oxidation under conditions where the PN pool
was kept fully reduced. This correlation was subsequently challenged by
the finding that the permeability transition induced by organic
hydroperoxides could be inhibited by butylhydroxytoluene while
glutathione remained oxidized (24) and by the observation that
oxidation of glutathione with 1,3-bis(2-chloroethyl)-1-nitrosourea
(which prevents PN oxidation by inhibiting glutathione reductase) was
not followed by Ca efflux due to a permeability
transition(25) .
The effect of several oxidative agents on
mitochondrial membrane permeability has recently been rationalized
within the framework of the MTP voltage
dependence(1, 26) . The apparent pore gating potential (i.e. the threshold voltage for pore opening) is shifted to
more negative levels by dithiol oxidation, resulting in a
higher probability of pore opening at physiological membrane
potentials(1) . Conversely, dithiol reduction or substitution
with NEM is accompanied by a shift of the apparent gating potential to
less negative
levels, resulting in a lower probability of
pore opening(1) . The dithiol responsible for these effects can
be selectively blocked by derivatization with MBM, which can therefore
be used as a probe for the pore agonists that act at this
site(27) .
Several open questions remain. Is oxidation of the MBM- and NEM-sensitive dithiol linked to changes in the oxidation-reduction state of mitochondrial PN and/or glutathione? Is there a role for PN and glutathione oxidation in regulation of the apparent MTP voltage dependence, and what is their relative contribution? In this paper we describe the properties of two distinct sites that affect the probability of MTP opening through oxidation-reduction reactions. The first site is blocked by NEM but not by MBM and appears to be in redox equilibrium with the PN pool. The second site is blocked by MBM, coincides with the oxidation-reduction-sensitive dithiol we have recently identified(1) , and is presumably in equilibrium with the glutathione pool. The existence of at least two oxidation-reduction-sensitive regulatory sites on the MTP in equilibrium with both PN and glutathione readily resolves many conflicting reports in the literature and allows a better description of the MTP response to oxidative stress under a variety of in vitro conditions.
Rat liver mitochondria were prepared as described(28) . The fraction of mitochondria permeabilized to sucrose by MTP opening and the membrane potential were determined as described in detail elsewhere ( (29) and (30) , respectively).
PN and glutathione levels were determined after
preincubation of mitochondria under the conditions specified in the
table legends. The sequence of the addition of Ca,
EGTA, inducers, and inhibitors was as shown for Fig. 1, while
rotenone, when present, was included from the beginning of the
experiments. Incubation conditions were identical to those of the
light-scattering experiments, except that the FCCP concentration was
200 nM. PN or glutathione determinations were carried out 1
min after the addition of FCCP. NADH and NADPH levels were determined
on alkaline extracts and GSH level was determined on acid extracts
based on coupled enzyme reactions exactly as described in (23) . For the glutathione measurements, the concentration of
mitochondria was increased up to 2 mg of protein
ml
with a corresponding increase of all reagents
(EGTA, Ca
, FCCP, CsA, AsO, AcAc, etc.). Since the
total thiol content determined with 5,5`-dithiobis(2-nitrobenzoic acid)
at 412 nm in the same extracts gave exactly the same values of the
direct glutathione measurements, the latter procedure was routinely
used.
Figure 1:
Effects of NEM, MBM, and BOH on AsO- or
AcAc-dependent MTP opening induced by FCCP. The incubation medium
contained 0.2 M sucrose, 10 mM Tris-Mops, pH 7.4, 5
mM succinate-Tris, 1 mM P-Tris, 10
µM EGTA-Tris, 2 µM rotenone (final volume, 2
ml; 25 °C). The experiments were started by the addition of 1 mg of
mitochondria (not shown). Where indicated (the first addition being
made exactly 1 min after mitochondria) the following additions were
made: 20 µM Ca
, 0.5 mM AcAc (panel A, traces b-e) or 0.5 mM AsO (panel B, traces b-e), 0.5 mM EGTA-Tris, and 40 nM FCCP. At the arrow marked I, the following additions were made (both panels, traces c-e only): 25 µM MBM (traces
c), 25 µM NEM (traces d), or 1 mM BOH (traces e). Ф, the fraction of mitochondria that
have opened the MTP.
All chemicals were of the highest purity commercially available. Incubation conditions and further experimental details are given in the figure legends.
Figure 3:
Effect of AcAc on MTP opening induced by
FCCP. The experiments were carried out exactly as shown in Fig. 1. After the addition of 10 µM Ca, the indicated concentrations of FCCP were
added in the absence (closed symbols) or presence (open
symbols) of 1 mM AcAc. Panel A shows the
dependence of MTP opening on the FCCP concentration, while in panel
B the data are replotted as a function of the membrane potential
as determined on parallel samples incubated in the presence of CsA to
prevent depolarization due to pore opening (panel B, inset).
Figure 4:
Effect of rotenone on MTP opening induced
by FCCP. The experiments were carried out exactly as shown in Fig. 1in the presence (closed symbols) or absence (open symbols) of 2 µM rotenone. After the
addition of 10 µM Ca, the indicated
concentrations of FCCP were added. Panel A shows the
dependence of MTP opening on the FCCP concentration, while in panel
B the data are replotted as a function of the membrane potential
as determined on parallel samples incubated in the presence of CsA (panel B, inset).
In the experiments of Fig. 1B, the shift of MTP response to depolarization was induced with AsO, a selective dithiol cross-linker (32) and well characterized MTP agonist(1, 27) . The addition of 40 nM FCCP was again followed by MTP opening, which could be counteracted by both NEM (panel B, trace d; cf. (1) ) and MBM (panel B, trace c; cf. (27) ). The novel finding of this experiment is that the effect of AsO (or of its analog PhAsO, not shown) could not be blocked by BOH (panel B, trace e), which instead blocked the effects of AcAc (compare panels A and B).
Fig. 2summarizes a series of experiments carried out as shown in Fig. 1, where the concentration dependence of the effects of MBM, BOH, and NEM was studied. The experiment documents that (i) MBM was not able to inhibit the effects of AcAc at concentrations that fully prevented the effects of AsO (Fig. 2A) (this was also the case with dithiothreitol, which did not inhibit the effects of AcAc at concentrations that fully reverted the effects of AsO (not shown, but see (1) )); (ii) that conversely, the effects of AsO were totally insensitive to concentrations of BOH that fully prevented the effects of AcAc (panel B); and (iii) that both the effects of AcAc and AsO were completely blocked by identical concentrations of NEM (panel C).
Figure 2: Concentration dependence of the effects of NEM, MBM, and BOH on AsO- or AcAc-dependent MTP opening induced by FCCP. The experiments were carried out exactly as described in Fig. 1, and the fraction of mitochondria with an open pore was determined after the addition of FCCP after treatment with 1 mM AcAc (closed symbols) or 1 mM AsO (open symbols) in the presence of the indicated concentrations of MBM (panel A), BOH (panel B), or NEM (panel C). The concentration of FCCP was 50 nM in the experiments of panels A and C, and 40 nM in those of panel B. A different mitochondrial preparation was used for the experiments of each panel. Since the fraction of mitochondria opening the MTP after the addition of FCCP in the presence of AcAc or AsO was nearly identical in all cases, the data were replotted as the percentage of the maximum for the sake of clarity, where 100% is the fraction of mitochondria that opened the pore in the presence of AcAc or AsO and in the absence of inhibitors.
To modulate the
oxidation-reduction state of PN with an independent method, in the
experiments of Fig. 4succinate-energized mitochondria were
incubated in the absence or presence of rotenone. It can be seen that
omission of rotenone (open symbols) led to a dramatic increase
of the population responding to FCCP relative to that observed in the
presence of rotenone (closed symbols). Also in this case the
response to uncoupler was identical in the absence or
presence of rotenone (panel B, inset), indicating
that rotenone omission caused a shift in the MTP gating profile (panel B). This rotenone-dependent shift could be largely
inhibited by NEM but not by MBM or dithiothreitol (not shown).
Superimposable results, not shown here, were obtained with
rolliniastatin-2, a site I inhibitor that is not structurally related
to rotenone(33) .
The MTP response to graded additions of FCCP is somewhat variable in different mitochondrial preparations (see (1) and (29) for a discussion). In these experiments we have therefore chosen FCCP concentrations giving maximal uncoupling. The following results give therefore an upper limit for PN or glutathione oxidation under the experimental conditions of Fig. 1Fig. 2Fig. 3Fig. 4. Table 1shows that the addition of FCCP in the presence of rotenone did not appreciably oxidize PN or glutathione. As expected, NADH and NADPH oxidation could be achieved by either the addition of AcAc, or the omission of rotenone, both conditions affecting the MTP response to depolarization (see Fig. 3and Fig. 4). On the other hand, under both conditions glutathione remained fully reduced, and the difference between reduction levels of PN and glutathione was particularly large in the case of rotenone omission. These data indicate that under our experimental conditions the PN oxidation-reduction state is related to the MTP gating profile independently of changes in the oxidation-reduction state of glutathione.
Table 1also shows that in the presence of rotenone and FCCP the addition of AsO, which under these conditions causes pore opening, was not accompanied by oxidation of either PN or glutathione. The experiment therefore indicates that the oxidation-reduction-sensitive dithiol (1) can affect the MTP gating profile independently of changes in the oxidation-reduction state of PN. It appears likely that the AsO-reactive dithiol is in equilibrium with glutathione and that the AsO-complexed species, which is equivalent to the disulfide for MTP regulation(1) , cannot be reduced by glutathione and becomes thus unavailable for its natural oxidation-reduction partner (see also Table 3).
In the experiments of Table 1CsA was included to prevent any secondary effects of MTP opening on reduced PN and glutathione levels. In the experiments of Table 2, CsA was omitted and MTP opening was induced by AsO plus FCCP (i.e. by a condition that does not affect the level of reduced PN when the MTP is blocked by CsA; Table 1). Under these conditions PN oxidation could be reproducibly observed, confirming earlier reports that PN oxidation may well follow rather than precede the permeability transition(22, 23, 34) . To rule out the possibility that CsA was preventing PN oxidation by mechanism(s) unrelated to its inhibitory effects on the pore, we have measured PN and glutathione levels in AsO- and FCCP-treated mitochondria in the absence of CsA, but under conditions where MTP opening was prevented by the addition of MBM, NEM, or EGTA. The results, also presented in Table 2, indicate that PN remained reduced irrespective of the method used to prevent pore opening. These data indicate that PN oxidation is more likely to be the consequence than the cause of MTP opening when dithiol cross-linkers like AsO or PhAsO are used as the agonists, at variance from a recent suggestion to the contrary(35) .
In this paper we have shown that the apparent MTP gating potential can be modulated by oxidation-reduction effectors at two sites that can be distinguished experimentally. One site (which we will now call the ``P site'') appears to be modulated through the oxidation-reduction state of PN even when glutathione is fully reduced, and it accounts for the effects of MTP agonists like AcAc (19) or duroquinone(37) . The P site can be blocked by NEM but not by MBM. The other site (which we will now call the ``S site'') coincides with the oxidation-reduction-sensitive dithiol(1) , and it can be activated by reaction with AsO or PhAsO even when the PN pool is fully reduced. The S site can be blocked by both NEM and MBM. It appears that oxidants like TBH and DIA can oxidize both PN and glutathione and thus affect both the P and S sites. Irrespective of the precise mechanism by which glutathione and PN affect the MTP, oxidative stress causes an increased probability of pore opening when the concentrations of reduced glutathione and PN decrease.
Our findings demonstrate that MTP opening is correlated to both the oxidation-reduction state of PN through the P site and (presumably) of glutathione through the S site. We would like to stress that there is no direct evidence here or in the literature that glutathione as such is involved in pore regulation. Indeed, many glutathione oxidants could also directly oxidize the dithiol S site at the same time. Furthermore, since PN and glutathione are in oxidation-reduction equilibrium through PN transhydrogenase, glutathione reductase, and possibly mitochondrial thioredoxin reductase (38) , reagents like TBH and DIA can lead to oxidation of both glutathione and PN, thus affecting both sites at the same time.
While the NEM- and MBM-sensitive S site coincides with the dithiol we recently identified(1, 27) , the NEM-sensitive P site remains chemically undefined. Indeed, although the NEM reactivity may suggest that a cysteine thiol group is involved, no other compound we tested among a variety of thiol reagents (including mersalyl and methyl methanethiosulfonate, data not shown) was able to block the P site.
In principle, NAD(P)H (we could not resolve which PN is
responsible, even if the data of Table 3would suggest a better
correlation with NADH levels) might affect the MTP as a reductant,
possibly through a voltage-sensing element that could also be reduced
by glutathione. However, it should be mentioned that the P site is
insensitive to reductants like dithiothreitol and -mercaptoethanol
(data not shown). An alternative possibility is that PN may directly
interact with the P site modulating the MTP response to voltage by an
allosteric mechanism, which is consistent with the NADH-specific
inhibitory effect on the MTP reported by Hunter and Haworth (5, 39) in deenergized mitochondria. An example of
regulation of a channel by PN can be found for the voltage-dependent
anion channel of the outer mitochondrial membrane, where NADH (but not
NAD
, NADPH, or NADP
) decreases the
permeability to ADP by a factor of 6(40) .
The concept that
the MTP is modulated by oxidating agents at two sites resolves
several standing problems. It is clear from the experiments presented
here that the PN and glutathione oxidation-reduction state does not
necessarily affect MTP opening in an all-or-nothing fashion. In other
words, pore opening may or may not follow PN and/or glutathione
oxidation, depending on additional factors like e.g. the
magnitude of the membrane potential, the Ca load, and
the level of endogenous adenine nucleotides. Yet, under oxidative
conditions the apparent gating potential is demonstrably shifted toward
the resting level ( Fig. 3and Fig. 4). This explains why
MTP opening (and the ensuing Ca
efflux) can be
dissociated from the oxidation-reduction state of PN and glutathione
and why an unequivocal correlation between MTP opening and
oxidation-reduction levels of either glutathione or PN alone could not
be established, even if it is widely accepted in the field that pore
operation is indeed affected by the mitochondrial redox
state(4) .
The voltage inactivation properties of the
/
K
channels can be modulated by glutathione
through a cysteinyl residue close to the N terminus. This residue can
be located on either the pore-forming
subunit in rapidly
inactivating (A-type) K
channels (42) or on the
regulatory
1 subunit, which confers the rapidly inactivating mode
to otherwise slowly inactivating K
channels(43) .
In either case, rapid inactivation may involve formation of a disulfide
with a second cysteinyl residue in an as yet unidentified channel
region(42, 43) . Also relevant to the present
discussion, it has been noted that K
channel
subunits
bear a striking resemblance (both in terms of primary sequence and of
secondary structural elements) to the superfamily of NAD(P)H-dependent
oxidoreductases, with conservation of the PN binding site, suggesting
that PN binding could be involved in K
channel
regulation(44) . It must be stressed that
oxidation-reduction-sensitive K
channels exist in Escherichia coli(45) , which bear sequence homology to
the mammalian and Drosophila
/
K
channels (46) . The MTP might have conserved structural
features of the archetypal K
channel and thus
accomplish modulation of its voltage-dependence by glutathione and PN
by similar mechanisms.