Rotenone Inhibits the Mitochondrial Permeability Transition-induced Cell Death in U937 and KB Cells

Abstract

The permeability transition pore (PTP) is a mitochondrial inner membrane Ca 2؉ -sensitive channel that plays a key role in different models of cell death. Because functional links between the PTP and the respiratory chain complex I have been reported, we have investigated the effects of rotenone on PTP regulation in U937 and KB cells. We show that rotenone was more potent than cyclosporin A at inhibiting Ca 2؉ -induced PTP opening in digitonin-permeabilized cells energized with succinate. Consistent with PTP regulation by electron flux through complex I, the effect of rotenone persisted after oxidation of pyridine nucleotides by duroquinone. tert-Butyl hydroperoxide induced PTP opening in intact cells (as shown by mitochondrial permeabilization to calcein and cobalt), as well as cytochrome c release and cell death. All these events were prevented by rotenone or cyclosporin A. These data demonstrate that respiratory chain complex I plays a key role in PTP regulation in vivo and confirm the importance of PTP opening in the commitment to cell death.Studies over the past few years have led to the recognition that, in addition to their established role in energy metabolism, mitochondria are main actors of cell death (see Refs. 1 and 2 for recent reviews). Mitochondrial proapoptotic factors such as cytochrome c, apoptosis-inducing factor, and smac/diablo are normally confined to the mitochondrial intermembrane space. Once released into the cytosol, cytochrome c binds to Apaf-1, which prompts activation of caspases in the presence of ATP or dATP (3, 4), and smac/diablo counteracts the inhibitory effect of inhibitor of apoptotic proteins on caspases activity (5, 6), whereas apoptosis-inducing factor activates nuclear endonucleases (7,8). The mechanisms by which the outer mitochondrial membrane becomes permeable to these proapoptotic proteins are not totally understood, but evidence suggests that two non-mutually exclusive pathways may be concerned (1, 2). One relies on outer membrane channel(s) involving Bcl-2 family proteins, whereas the other is due to outer membrane rupture secondary to mitochondrial swelling and implicates an inner membrane channel, the permeability transition pore (PTP). 1 PTP opening in vitro leads to collapse of the proton-motive force, disruption of ionic homeostasis, mitochondrial swelling, and release of intermembrane space proteins, especially cytochrome c (9 -11). Involvement of the PTP in the commitment to cell death is supported by a large body of evidence based on the protective effect of two PTP inhibitors, namely cyclosporin A (CsA) (reviewed in Refs. 12-15) and bongkrekic acid (BA) (e.g. see , in several models of cell death. PTP opening in vivo is commonly appreciated as a CsA-sensitive mitochondrial depolarization but can also be visualized directly by fluorescence imaging using CsA-sensitive calcein diffusion through the mitochondrial inner membrane (22)(23)(24)(25).PTP regulation has been studied extensively in rat liver mitochondria. Although it is generally assumed that there is ...

Potential threat of Chlorpyrifos to human liver cells via the caspase-dependent mitochondrial pathways

“…The results of the fluorescence microscopy experiments show CPF can induce the disruption of MMP in a concentration-dependent manner (Figure 3(B)). When the MMP reduced, cytochrome c is released from mitochondria into the cytosol (Chauvin et al, 2001). Cytochrome c, a basic protein, is anchored in the inner mitochondrial membrane, and initiates caspase activation when released from the mitochondrial during apoptosis (Liu, Kim, Yang, Jemmerson, & Wang, 1996).…”

Coenzyme Q10 Prevents Apoptosis by Inhibiting Mitochondrial Depolarization Independently of Its Free Radical Scavenging Property

“…These considerations prompted us to hypothesize that what was observed with ubiquinone analogues applied to isolated mitochondria (19 -24) could underlie a natural phenomenon operating in the whole cell. In line with this possibility, the respiratory chain complex I has been suggested to be a constituent of mitochondrial PTP (26). Armstrong and co-workers (27,28) have designed a new model for mitochondrial PTP, in which the ROS producer respiratory chain complex III is functionally linked to the permeability transition.…”

“…On the basis of these considerations, CoQ 10 may be a candidate as a functional analogue of the Bcl-2 protein (63). Moreover, association of complexes I and III of the mitochondrial respiratory chain with mitochondrial PTP (19,20,(23)(24)(25)(26)(27)(28) and the integration of CoQ 10 in both complexes suggest the role of CoQ 10 as a structural element and modulator of mitochondrial PTP. Experiments are now in progress to obtain direct evidence of this possibility and to "pin-point" localization of CoQ 10 in the PTP complex.…”

The Characterization of Mitochondrial Permeability Transition in Clonal Pancreatic β-Cells

“…Brain mitochondria demonstrate high resistance to MPT opening (10), low sensitivity to cyclosporin A (5), different responses to MPT modifiers, and a distinctive composition of Ca 2ϩ -phosphate complexes sequestered in the matrix (11). Mitochondria from skeletal muscle, unlike hepatocyte mitochondria, show the dependence of MPT on electron flux through respiratory complex 1 (12). Most importantly, basal endogenous MPT activity (transient pore opening), which affects Ca 2ϩ signaling, oxidative phosphorylation, and ROS production, is different in different cell types, which can be attributed to the variable cellular redox states (13).…”