(Received for publication, March 5, 1997, and in revised form, May 23, 1997)
From the We have studied the effects of singlet oxygen
produced by photodynamic action on the cyclosporin A-sensitive
permeability transition (PT) in isolated rat liver mitochondria.
Mitochondria were incubated with 3 µM
hematoporphyrin and irradiated at 365 nm with a fluence rate of 25 watts/m2. For short durations of irradiation (60 s) the
adenine nucleotide translocase was inactivated, but mitochondria
retained their ability to form a proton electrochemical gradient and
accumulated Ca2+ and Pi at the same rate as
non-irradiated controls. Strikingly, however, the oxidative effects of
photodynamic action prevented opening of the PT pore which is normally
induced by Ca2+ plus Pi or by treatment with
diethyl pyrocarbonate (a histidine reagent) or diamide (a thiol
oxidant). We show that the most likely targets for photodynamic action
are critical histidines that undergo degradation. Irradiated,
hematoporphyrin-loaded mitochondria treated with diethyl pyrocarbonate
or diamide still undergo the PT when treated with phenylarsine oxide,
which reacts with a critical dithiol involved in pore modulation
(Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti,
S., and Bernardi, P. (1994) J. Biol. Chem. 269, 16638-16642). These data suggest (i) that the dithiol cysteines are
not oxidized by photodynamic action, but rather became inaccessible to
oxidants; and (ii) that irradiation of hematoporphyrin-loaded
mitochondria does not lead to pore denaturation, but rather to
site-selective inactivation of discrete pore functional domains.
When Ca2+-loaded mitochondria are treated with a
variety of inducing agents, the opening of
Ca2+-dependent pores leads to the so-called
permeability transition of the inner membrane, which is specifically
inhibited by nanomolar concentrations of cyclosporin A. Among
Ca2+-dependent pore inducers, oxidizing agents
have received considerable attention, and redox state changes of
pyridine nucleotides, glutathione, or sulfhydryl groups have been shown
to have a prominent role in the mechanism (or in the control) of
Ca2+ efflux following pore opening (for general reviews,
see Refs. 1 and 2).
In the present work, we have studied the effects of the very reactive
species, singlet oxygen, 1O2, on isolated rat
liver mitochondria. The presence of 1O2 as a
transient species able to damage cells, mainly at the mitochondrial
level, can be observed in disease, such as in porphyria (3), and in the
treatment of solid tumor by porphyrin photodynamic therapy (4). The
effects of 1O2 on substrate transport,
respiration, and phosphorylation are well documented, but few reports
deal with those on the uptake and release of Ca2+ from
mitochondria (for a general review, see Ref. 5). In 1981 we have
reported (6) that treatment with 1O2 produced
by porphyrin photodynamic action can prevent Ca2+ release
due to membrane "damage" resulting from massive Ca2+
loading. At that time, our interpretation was that such a release from
Ca2+-overloaded mitochondria was probably of no
physiological relevance. This aspect must now be reconsidered in the
light of recent developments in the mitochondrial
PT1 field. It is now widely
accepted that Ca2+ release can occur through opening of a
specific channel sensitive to cyclosporin A rather than to unspecific
membrane damage. These advances prompted us to study more thoroughly
the effect of photodynamic action on mitochondrial energy transduction
and Ca2+ fluxes. As in our previous studies, HP has been
chosen as an exogenous photosensitizer in isolated rat liver
mitochondria. Our results show that, in sharp contrast with other
activated oxygen species such as H2O2 or
O Photoirradiated, HP-loaded mitochondria treated with DEPC or DIA still
undergo the PT when treated with PhAsO, which reacts with a critical
dithiol involved in pore modulation (7). These data indicate that the
dithiol cysteines are not oxidized by 1O2, but
rather became inaccessible to oxidants, and suggest that irradiation of
HP-loaded mitochondria does not lead to pore denaturation, but rather
to site-selective inactivation of discrete pore functional domains
comprising critical histidines.
Preparation of rat liver mitochondria was performed as described
previously (8). To compare with maximum accuracy the various responses,
mitochondria were placed in a vessel fitted with both an oxygen and a
Ca2+ electrode (9), and all the irradiations were performed
under the same optical conditions, i.e. 3 µM
HP, giving an absorbance of 0.14/cm at the irradiation wavelength of
365 nm. Irradiation was performed using a Philips HPW 125-watt lamp
(Philips, Eindhoven, The Netherlands) after 120 s incubation in
the dark, since uptake of HP in isolated mitochondria reaches a plateau
within 120 s (10). The fluence rate incident to the
mitochondrial suspension was 25 watts/m2, as measured with
a Black Ray UV meter (J 221, Ultraviolet Products, Inc., San Gabriel,
CA). All irradiations were performed at 25 °C under magnetic
stirring. Proper controls were carried out, indicating that the
inhibitors used (rotenone, oligomycin, and cyclosporin A) gave a
negligible light absorption at the wavelength of irradiation and that
neither photosensitizer addition in the dark nor illumination alone
were able to produce any change in the measured parameters (results not
shown).
Pi carrier activity was followed using the classical
swelling technique of Chappel and Haarhoff (11).
Measurement of ADP/ATP exchange was performed fluorometrically in
control and photosensitized mitochondria as described by Passarella
et al. (12).
Mitochondrial swelling was followed spectroscopically as the decrease
in the absorbance of the mitochondrial suspension at 540 nm measured
with a SLM Aminco DW 2000 spectrophotometer operated in the split beam
mode (13).
All reagents were of the finest available grade and were used without
further purification. HP was purchased from Aldrich and 1 mM stock solutions were prepared in dimethyl sulfoxide. Cyclosporin A, a gift from Dr. A. Roche of the Laboratoires Sandoz, Rueil Malmaison, France, was dissolved in ethanol. Incubation conditions and further experimental details are given in the figure legends.
We have long been interested in the
relative effects of irradiation on specific mitochondrial functions. We
have shown that at increasing doses of radiation, oxidative
phosphorylation is the first function to be lost, whereas respiration
and Ca2+ cycling are more resistant (9). These results
suggest that selective targets for the photodynamic effect of
porphyrins exist in mitochondria and that these may be exploited to
understand the mechanism(s) of phototoxicity.
Fig. 1 shows the effects of increasing
durations of irradiation in the presence of 3 µM HP on
respiration in state 4 (in the absence of ADP) or 3 (in the presence of
ADP) and after addition of FCCP. Short exposures to irradiation (up to
60 s) did not affect state 4 respiration, and ADP-stimulated
respiration was more sensitive to photodynamic action than
FCCP-uncoupled respiration. This suggests that neither electron
transport nor membrane potential is severely impaired after short
durations of irradiation, in good agreement with Ref. 14. Fig.
2 shows that in fact damage to the AdNT
(rather than damage to the ATP-synthase, as indicated before in Ref.
14) is responsible for the inhibition of the oxidative phosphorylation. Indeed, the exchange rate as a function of ADP concentration at increasing irradiation times indicates a strong non-competitive inhibition of the carrier. From this set of experiments, we conclude that the AdNT is a sensitive target that is easily inactivated by HP
photodynamic action.
As indicated above, Ca2+ cycling in isolated
mitochondria appears to be less sensitive to photodynamic action than
oxidative phosphorylation (9). In this paper, we have specifically
addressed the issue of how irradiation affects Ca2+ uptake
and release from mitochondria and the pathways for Ca2+
efflux under these conditions.
To study the effects of irradiation on Ca2+ transport,
Ca2+ uptake was measured after various periods of
irradiation and recorded as the initial rate of Ca2+ influx
in mitochondria energized with succinate + rotenone under conditions
where Ca2+ uptake is not limited by the rate of respiration
(20 µM CaCl2 in the presence of 10 mM Mg2+, see Ref. 15). Fig.
3 shows that the initial rate of
Ca2+ uptake strongly declined only after 180 s of
irradiation. This is consistent with the results of Fig. 1, indicating
that brief irradiations leave mitochondria with a good respiratory
capacity, used here to drive Ca2+ uptake along the
electrochemical gradient. Inhibition of Ca2+ transport was
not a consequence of inactivation of the Pi carrier, since
up to 240 s of irradiation had negligible effects on the rate of
Pi uptake measured according to Ref. 11 (data not
shown).
Fig. 4 shows a typical experiment where
mitochondria energized with succinate + rotenone, and loaded with 80 µM Ca2+, were subsequently exposed to
continuous irradiation. Initially, mitochondria treated in this way
remained in a steady state, maintaining an external Ca2+
concentration of about 1 µM. After over 120 s of
irradiation, a process of Ca2+ efflux ensued, which could
not be inhibited by the addition of 0.8 µM cyclosporin A
to the medium. We conclude that long durations of irradiation (>120 s)
inhibit respiration to the point that the proton electrochemical
gradient can no longer be maintained. Under these conditions
Ca2+ leaves the matrix by reverse operation of the
uniporter rather than through the PT. Shorter irradiation times, on the
other hand, do not impair Ca2+ uptake or retention by
mitochondria (Figs. 3 and 4). The latter condition was then exploited
to test the effect(s) of photodynamic action on the PT.
The experiment of Fig. 5
illustrates the effect of increasing irradiation duration on the
ability of HP-treated mitochondria to take up and retain
Ca2+. Non-irradiated mitochondria did not retain a 150 µM Ca2+ pulse, and a fast process of
Ca2+ release readily followed the initial phase of
Ca2+ uptake (trace A) because of a PT (see Fig.
6). After 30 s of irradiation,
mitochondria readily accumulated the initial 150 µM
Ca2+ pulse (trace B), whereas Ca2+
efflux was triggered by a further 20 µM Ca2+
load (trace B). After 60 s of irradiation the ability
of mitochondria to retain Ca2+ was further improved
(trace C), whereas higher irradiation periods impaired the
initial rate of Ca2+ uptake (trace D), as
expected from the extent of respiratory inhibition and therefore of
impairment of membrane potential regeneration (Figs. 3 and 4). For this
reason, 60 s of irradiation were used in all subsequent
experiments.
The experiments of Fig. 6 were designed to test the pathways for
Ca2+ efflux (followed by a Ca2+-selective
electrode) and the occurrence of a PT (followed spectroscopically) in
sucrose-based media. Fig. 6, panel A, shows that
non-irradiated mitochondria underwent a fast, spontaneous
Ca2+ release process when challenged with a 150 µM Ca2+ pulse (trace a), which
could be totally prevented by 800 nM cyclosporin A (not
shown). As expected, spontaneous Ca2+ efflux was prevented
by 60 s of irradiation, yet Ca2+ efflux was readily
observed after the addition of the uncoupler FCCP (trace b).
This process of Ca2+ efflux was not prevented by
cyclosporin A (trace c), indicating that upon addition of
FCCP rapid reversal of the uniport takes place. The experiments
reported in Fig. 6, panel B, show that non-irradiated
mitochondria were permeabilized to sucrose by Ca2+
(trace a), whereas irradiated mitochondria maintained their
permeability barrier when challenged with an identical Ca2+
load (trace b), and yet underwent permeabilization and
swelling upon addition of FCCP (trace b), in a process that
maintained full sensitivity to cyclosporin A (trace c).
These findings represent a first indication that the PT pores can still
form and open upon depolarization even in photoirradiated
mitochondria.
Reversible protonation of histidyl residues and oxidoreduction of thiol
groups play an important role in the modulation of pore opening (2). As
1O2 is very reactive with histidine and thiol
groups (16), we have determined whether inducers specifically reacting
with these structures have a different mode of action after
irradiation. Using the same swelling technique, PT was studied in
mitochondria loaded with 40 µM Ca2+.
Fig. 7, panel A, shows that
addition of DIA to control mitochondria induced the expected PT, with
rapid permeabilization to sucrose after a short lag phase (trace
a). Strikingly, however, DIA was not able to cause pore opening in
irradiated mitochondria (trace b), suggesting that the pore
can no longer be induced by thiol oxidation. In irradiated mitochondria
the pore could still be opened by the hydrophobic dithiol reagent,
PhAsO (trace c), indicating that the dithiol cysteines have
not undergone oxidation but are rather not accessible to oxidants from
the aqueous phase. It should be mentioned that addition of PhAsO alone
in these protocols was followed by pore opening (not shown). Finally,
the lack of pore opening by DIA could be observed in a range of
concentrations that are very effective at pore opening in control
mitochondria (Fig. 7, panel B).
Fig. 8, panel A, reports the
results of a similar experiment where control or irradiated
mitochondria were treated with DEPC. As expected (17) DEPC treatment
caused pore opening in control mitochondria (trace a). On
the other hand, DEPC was unable to cause a PT in irradiated
mitochondria (trace b), suggesting that histidine(s) are
among the targets of 1O2. Also in this case the
pore remained competent for opening, as demonstrated by the rapid
permeabilization following the addition of PhAsO (trace c).
Fig. 8, panel B, shows the effects of increasing concentrations of DEPC on pore opening in control (closed
symbols) and irradiated mitochondria (open symbols). It
can be appreciated that at concentrations higher than 0.2 mM DEPC the pore slowly opened even in irradiated
mitochondria. This effect, however, can be easily accounted for by
respiratory inhibition, which in turn causes pore opening because of
membrane depolarization (not shown, but see Ref. 17).
We have described the effects of
irradiation on the mitochondrial PT in HP-treated mitochondria, a
well-defined system where the highly reactive species,
1O2 is generated. We have shown that
irradiation does not cause pore opening. Rather, irradiated
HP-treated mitochondria no longer undergo a PT when challenged with
Ca2+ plus Pi, DIA, or DEPC (Figs. 6, 7, 8). The
experimental conditions (HP concentration, wavelength of irradiation,
power density, duration of irradiation, and Ca2+ load) were
carefully selected to minimize the effects of irradiation on the basic
parameters modulating pore activity. In particular, (i) the respiratory
capacity and the coupling efficiency of irradiated mitochondria were
sufficient to form and maintain a proton electrochemical gradient (see
also Ref. 18), as indicated by the maintenance of respiratory control
with FCCP (Fig. 1); furthermore, a decreased proton electrochemical
gradient should have promoted rather than inhibited the PT
(2) and cannot therefore account for the present observations of pore
inactivation. (ii) The steady-state Ca2+ accumulation
maintained by irradiated mitochondria was identical to that maintained
by the controls (Figs. 3 and 4), and the Pi carrier was
negligibly affected by irradiation in the presence of HP (not shown);
thus, pore inhibition cannot be explained by a lower Ca2+
load nor by a different response of matrix pH to Ca2+
uptake and release because of impaired Pi equilibration.
(iii) Even if we cannot exclude a minute generation of O 1O2 is a very
reactive, transient species capable of damaging nucleic acids,
proteins, and lipids (16). Its lifetime is very short in biological
materials, which restricts its action to the close vicinity (<0.1
µm) of the photosensitizer (24). Although the membrane lipid phase is
clearly involved in modulating the PT, possibly through surface
potential effects (25, 26), we tend to exclude that pore inactivation
by HP photodynamic action is due to lipid peroxidation, which should
rather promote the PT (1).
The most likely target for
1O2-dependent pore inhibition
appears to be proteinaceous in nature. HP dissolved in aqueous media preferentially accumulates in protein-binding sites of the inner mitochondrial membrane (27). Photosensitized protein damage can produce
important primary structural and functional changes through the
photo-oxidation of five amino acid residues, namely cysteine,
histidine, methionine, tryptophan, and tyrosine (16). Two of these
residues readily oxidized by 1O2, cysteine and
histidine, have been shown to play a critical role in regulation of the
PT (7, 17).
Dithiol-disulfide interconversions at vicinal cysteines affect the pore
open-closed transitions, a higher open probability being associated
with the disulfide (7). The actual dithiol oxidant appears to be
oxidized glutathione, which would mediate pore opening by a variety of
oxidants (23). Cysteine is oxidized to cystine by
1O2 (16), and in the context of pore regulation
by dithiols one could have predicted an increased probability of pore
opening rather than the observed pore inactivation. These findings
indicate that the inactivating effects of 1O2
cannot be simply ascribed to cysteine oxidation leading to a
dithiol-disulfide interconversion. The observed pore inactivation probably involves different SH groups or is the consequence of a
conformational change brought about by photooxidation of other residues, which in turn prevents access of oxidized glutathione to the
dithiol. This is supported by the finding that DIA, an SH group
oxidant, is no longer effective at inducing pore opening in irradiated,
HP-treated mitochondria; yet, pore opening under these conditions can
still be observed by addition of the hydrophobic reagent PhAsO (Fig.
7), which reacts with vicinal dithiols to form a stable complex. Since
pore opening by DIA and PhAsO is inhibited by
N-ethylmaleimide and monobromobimane with the same apparent
I50 (7, 28), we conclude that their site of action coincides, and we deduce that critical dithiols have not undergone oxidation as a result of irradiation.
The pore open-closed transitions are also regulated by reversible
protonation of histidine residues from the matrix side of the membrane,
with reversible pore closure at acidic matrix pH values that can be
prevented by histidine carbethoxylation with DEPC (17). Free histidine
residues can be photodegraded to several different compounds after
cycloaddition of 1O2 (29). Based on the
inactivating effects of irradiation on the pore, one can speculate that
histidine degradation following cleavage of the imidazole ring by
1O2 may suppress the regulation of pore opening
which depends on histidine protonation-deprotonation. Finally, our
findings suggest that under normal conditions the role of histidines is
in fact to allow rather than prevent opening of the pore, consistent
with the optimum matrix pH for pore opening at 7.3 (17),
i.e. a pH value at which histidines are largely
deprotonated.
Among the identified mitochondrial targets for
1O2 the AdNT stands out for its extreme
sensitivity to photoinactivation (14), which can be traced to thiol
group oxidation at sites critical for transport (30). Since the pore is
affected by inhibitors of the AdNT, it has long been suggested that the
translocase is involved, directly or indirectly, in pore formation or
modulation (see Refs. 1, 2, and 25 for discussion). More direct
evidence has recently come with the demonstrations (i) that the AdNT
reconstituted in giant liposomes exhibits a striking
Ca2+-dependent, high-conductance channel
activity with a marked voltage dependence (31) reminiscent of pore
behavior both in isolated mitochondria (7) and in single channel
measurements on mitoplasts (32); and (ii) that complexes enriched in
hexokinase, porin, and the AdNT exhibit
Ca2+-dependent and cyclosporin A-sensitive
high-conductance channel activity in planar lipid bilayers (33). While
it is tempting to speculate that photoinactivation of the AdNT might be
responsible for inactivation of the pore as well, we think that
evidence remains circumstantial and that photoinactivation might well
hit more than one critical target, including pore regulatory proteins
like mitochondrial cyclophilin (34, 35).
In conclusion, the data presented in this paper
support the view that photodynamic action leads to pore inactivation
because of selective interference with critical histidine(s), rather
than because of a generic pore denaturation, and suggest that the toxic consequences of the photodynamic effect in porphyrias and related disorders may involve an impairment of mitochondrial function also
through inactivation of the pore, which is as sensitive to irradiation
as is the AdNT. A thorough characterization of the complex consequences
of histidine photooxidation on a variety of pore regulatory features
(like modulation by the membrane potential and by matrix pH) is
currently under way in our laboratories.
We are very grateful to F. Vinzens for
excellent technical assistance.
Laboratoires de Biophysique et de
Photobiologie, INSERM U 201 et CNRS URA 481, Muséum National
d'Histoire Naturelle, 43 rue Cuvier,
75231 Paris Cédex 05, France and the ¶ Consiglio Nazionale
delle Ricerche,
Centro Biomembrane,
2 (1), 1O2 does not induce PT. Rather
HP photodynamic action leads to suppression of the PT induced by
Ca2+ plus Pi, DEPC, or DIA.
Effects of HP Photodynamic Action on Respiration and AdNT
Translocator Activity
Fig. 1.
Effect of HP + light on respiration.
Mitochondria (0.5 mg/ml) were suspended in a medium containing 24 mM glycylglycine, 10 mM MgCl2, 60 mM KCl, 7 mM KH2PO4, 87 mM sucrose, pH 7.4, 14 mM succinate, and 2 µg/ml rotenone) in the presence of 3 µM HP in a
thermostated (T = 25 °C) water-jacketed vessel. The
O2 consumption was followed using a Clark electrode
connected to a recorder. After 120 s incubation, the suspension
was irradiated. Respiration of mitochondria in state 4 (), in state
3 (
) (1 mM ADP), or uncoupled (
) (1.30 µM FCCP) is plotted versus the duration of irradiation.
[View Larger Version of this Image (16K GIF file)]
Fig. 2.
Effects of HP + light on the AdNT
activity. Experimental conditions were as in Fig. 1. After
irradiation for various times, measurement of ADP/ATP efflux was
performed in control mitochondria (), and in mitochondria irradiated
for 60 s (
), 120 s (
), or 240 s (×) using an
ATP-detecting system, exactly as described in Ref. 12.
[View Larger Version of this Image (16K GIF file)]
Fig. 3.
Effects of HP + light on the rate of
Ca2+ influx in mitochondria. Experimental conditions
were as in Fig. 1. Ca2+ movements were followed using a
Ca2+-selective electrode (Radiometer F 2110 calcium
selectrode, Copenhagen, Denmark) inserted into the reaction chamber.
After irradiation for various times, 20 µM
Ca2+ was added to the medium. Ca2+ uptake was
measured as the initial rate of Ca2+ influx in
nmol/mg/min.
[View Larger Version of this Image (13K GIF file)]
Fig. 4.
Effects of HP + light on mitochondria loaded
with Ca2+ before irradiation. Experimental conditions
were as in Fig. 3. Mitochondria (m) were loaded with 80 µM Ca2+ and continuously irradiated
(h). The same trace was obtained in the presence of 0.8 µM cyclosporin (data not shown). Dashed trace
represents non-irradiated mitochondria.
[View Larger Version of this Image (9K GIF file)]
Fig. 5.
Ca2+ loading of mitochondria
following a preliminary irradiation with various doses of light.
Experimental conditions were as in Fig. 3. Non-irradiated mitochondria
loaded with 150 µM Ca2+ (arrows)
(A) and mitochondria irradiated for 30 s
(B), 60 s (C) or 90 s (D)
were loaded immediately after irradiation with 150 µM
Ca2+ (arrows), followed by additions of 20 µM Ca2+ where indicated
(arrows).
[View Larger Version of this Image (14K GIF file)]
Fig. 6.
Effects of HP + light on mitochondrial
Ca2+ fluxes and on permeabilization to sucrose. The
incubation medium contained 200 mM sucrose, 10 mM Tris-Mops, pH 7.4, 5 mM succinate Tris, 1 mM Pi, 10 µM EGTA-Tris, 2 µg/ml
rotenone, and 3 µg/ml oligomycin. Panel A,
Ca2+ efflux measured with a Ca2+-selective
electrode. Where indicated (arrows) 0.5 mg/ml mitochondria (m), 150 µM Ca2+, and 0.2 µM FCCP were added. Trace a, control
mitochondria; trace b, mitochondria irradiated for 60 s
(h) in the presence of 3 µM HP before
Ca2+ loading; trace c, mitochondria irradiated
as in trace b in the presence of 0.8 µM
cyclosporin A. Panel B, PT formation followed spectroscopically. The experiments were started by addition of 0.5 mg/ml mitochondria (not shown). Trace a, control
mitochondria; trace b, mitochondria irradiated for 60 s
(h
) in the presence of 3 µM HP before
Ca2+ loading; trace c, control and mitochondria
irradiated as in trace b in the presence of 0.8 µM cyclosporin A. Where indicated (arrows), 150 µM Ca2+ and 0.2 µM FCCP
were added.
[View Larger Version of this Image (10K GIF file)]
Fig. 7.
Effect of DIA on PT in mitochondria treated
with HP + light. Experimental conditions are as in Fig. 6,
panel B. Panel A: trace a, control mitochondria;
traces b and c, mitochondria irradiated for
60 s in the presence of 3 µM HP. Where indicated (arrows) 40 µM Ca2+, 100 µM DIA, and (trace c only) 10 µM
PhAsO were added. Panel B, rate of mitochondrial
permeabilization (A/min) as a function of the
concentration of DIA under the same experimental conditions shown in
panel A. Open symbols, irradiated mitochondria;
closed symbols, non-irradiated mitochondria.
[View Larger Version of this Image (10K GIF file)]
Fig. 8.
Effect of DEPC on PT in mitochondria treated
with HP + light. Experimental conditions were as in Fig. 6,
panel B, except that Pi was omitted. Panel
A: trace a, control mitochondria; traces b and
c, mitochondria irradiated for 60 s in the presence of
3 µM HP. Where indicated (arrows) 40 µM Ca2+, 200 µM DEPC, and
(trace c only) 10 µM PhAsO were added.
Panel B, rate of mitochondrial permeabilization
(A/min) as a function of the concentration of DEPC under
the same experimental conditions shown in panel A. Open
symbols, irradiated mitochondria; closed symbols,
non-irradiated mitochondria.
[View Larger Version of this Image (10K GIF file)]
General Considerations
2 due
to either energy transfers from HP excited triplet state (19) or
increases in the respiratory chain e
leakage
after irradiation (20), ample evidence (reviewed in Refs. 1 and 2)
indicates that reactive oxygen species promote the PT, most
likely through oxidation of matrix glutathione (21-23); therefore, it
is unlikely that pore inhibition is mediated by other activated oxygen
species, which would rather counteract the inactivating effects of
1O2. From these considerations we conclude that
pore inhibition in irradiated, HP-treated mitochondria most likely
depends on direct effects of 1O2 on
critical residues important for pore function. Irrespective of the
detailed mechanism(s) by which irradiation interferes with the pore,
our data are consistent with the idea that 1O2
selectively inactivates specific domains, either on the pore itself or
on pore regulatory protein(s), normally acting as sensors for
modulatory signals. Indeed, the effects of irradiation cannot be
ascribed to a denaturation of the pore, since a PT can still be
observed in irradiated mitochondria upon addition of FCCP after Ca2+ plus Pi loading (Fig. 6) or upon addition
of PhAsO after treatment with DIA or with DEPC (Figs. 7 and 8).
*
This research was supported in part by a grant from the
European Union through the HCM program, "PDT Euronet" ERBCHRXCT
930178, and by CNR Grant 102006.04.9305026/27 (Italy-France cooperative scientific program).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: Laboratoires de
Biophysique et de Photobiologie, INSERM U 201 et CNRS URA 481, Muséum National d'Histoire Naturelle, 43 rue Cuvier, 75231 Paris Cédex 05, France. Tel.: 33 1-40793691; Fax: 33 1-40793705.
1
The abbreviations used are: PT, permeability
transition; HP, hematoporphyrin IX; DEPC, diethyl pyrocarbonate; DIA,
diamide; AdNT, adenine nucleotide translocase; PhAsO, phenylarsine
oxide; FCCP, carbonylcyanide-p-trifluoromethoxyphenyl
hydrazone; Mops, 4-morpholinepropanesulfonic acid.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.