Singlet Oxygen Produced by Photodynamic Action Causes Inactivation of the Mitochondrial Permeability Transition Pore

Abstract

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 M hematoporphyrin and irradiated at 365 nm with a fluence rate of 25 watts/m 2 . 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 Ca 2؉ and P i 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 Ca 2؉ plus P i 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, hematoporphyrinloaded 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 Ca 2ϩ -loaded mitochondria are treated with a variety of inducing agents, the opening of Ca 2ϩ -dependent pores leads to the so-called permeability transition of the inner membrane, which is specifically inhibited by nanomolar concentrations of cyclosporin A. Among Ca 2ϩ -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 Ca 2ϩ 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, 1 O 2 , on isolated rat liver mitochondria. The presence of 1 O 2 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 1 O 2 on substrate transport, respiration, and phosphorylation are well documented, but few reports deal with those on the uptake and release of Ca 2ϩ from mitochondria (for a general review, see Ref. 5). In 1981 we have reported (6) that treatment with 1 O 2 produced by porphyrin photodynamic action can prevent Ca 2ϩ release due to membrane "damage" resulting from massive Ca 2ϩ loading. At that time, our interpretation was that such a release from Ca 2ϩ -overloaded mitochondria was probably of no physiological relevance....

Targeting PBR by Hexaminolevulinate-Mediated Photodynamic Therapy Induces Apoptosis through Translocation of Apoptosis-Inducing Factor in Human Leukemia Cells

“…Thus, it seems that the loss of DW m is responsible for the AIF translocation, whereas the release of cytochrome c is not strictly dependent on DW m (37)(38)(39). Recent studies have suggested that the components of the permeability transition pore work together with proteins of Bcl-2 family to control the permeability transition pore opening in a specific manner for the release of mitochondrial proapoptotic proteins (39)(40)(41)(42). Two sites that help control the permeability transition pore have been proposed, the S and the P sites (40)(41)(42).…”

“…Two sites that help control the permeability transition pore have been proposed, the S and the P sites (40)(41)(42). The S site, which is cyclosporin A or bongkrekic acid sensitive (41), is responsible for the release of AIF, whereas the P site, which is cyclosporine A insensitive, is involved in the release of cytochrome c.…”

“…Recent studies have suggested that the components of the permeability transition pore work together with proteins of Bcl-2 family to control the permeability transition pore opening in a specific manner for the release of mitochondrial proapoptotic proteins (39)(40)(41)(42). Two sites that help control the permeability transition pore have been proposed, the S and the P sites (40)(41)(42). The S site, which is cyclosporin A or bongkrekic acid sensitive (41), is responsible for the release of AIF, whereas the P site, which is cyclosporine A insensitive, is involved in the release of cytochrome c.…”

Photodynamic action of porphyrin on Ca2+ influx in endoplasmic reticulum: a comparison with mitochondria

“…Inhibition of Ca# + uptake is obtained after the collapse of the electric field built up by respiratory chain proton pumps or ATP hydrolysis. However, it has been reported [6,37] that photodamage of the electron transport chain is not a primary event after photodynamic action. Irradiation with increasing doses of light in the presence of HP produces first an inhibition of oxidative phosphorylation followed by an inhibition of Ca# + uptake and finally an arrest of respiration.…”

ROS: Reactive Oxygen Species

“…EVects on the bioenergetics, (Atlante et al 1989;Moreno 1981, 1990) primarily on the mitochondrial permeability transition pore (Salet et al 1997) have been investigated in detail (Dummin et al 1997;Morgan and OseroV 2001;Plaetzer et al 2002). PDT may induce permeabilization of both mitochondrial membranes, dissipation of the transmembrane potential, and a release of apoptosis-related proteins (such as cytochrome c, apoptosis inducing factor, second mitochondria derived activator of caspases and certain procaspases) into the intermembrane space (Almeida et al 2004;Oleinick et al 2002).…”