Alloxan-induced Mitochondrial Permeability Transition Triggered by Calcium, Thiol Oxidation, and Matrix ATP

Abstract

In addition to their critical function in energy metabolism, mitochondria contain a permeability transition pore, which is regulated by adenine nucleotides. We investigated conditions required for ATP to induce a permeability transition in mammalian mitochondria. Mitochondrial swelling associated with mitochondria permeability transition (MPT) was initiated by adding succinate to a rat liver mitochondrial suspension containing alloxan, a diabetogenic agent. If alloxan was added immediately with or 5 min after adding succinate, MPT was strikingly decreased. MPT induced by alloxan was inhibited by EGTA and several agents causing thiol oxidation, suggesting that alloxan leads to permeability transition through a mechanism dependent on Ca 2؉ uptake and sulfhydryl oxidation. Antimycin A and cyanide, inhibitors of electron transfer, carbonyl cyanide m-chlorophenylhydrazone, and oligomycin all inhibited MPT. During incubation with succinate, alloxan depleted ATP in mitochondria after an initial transient increase. However, in a mitochondrial suspension containing EGTA, ATP significantly increased in the presence of alloxan to a level greater than that of the control. These results suggest the involvement of energized transport of Ca 2؉ in the MPT initiation. Addition of exogenous ATP, however, did not trigger MPT in the presence of alloxan and had no effect on MPT induced by alloxan. We conclude that alloxan-induced MPT requires mitochondrial energization, oxidation of protein thiols, and matrix ATP to promote energized uptake of Ca 2؉ .Mitochondrial permeability transition (MPT) 1 is associated with an increase in the permeability of the mitochondrial inner membrane to solutes with a molecular mass of 1.5 kDa or lower and can disturb mitochondrial functions in several ways (1-3). It is generally accepted that MPT is due to the opening of a so-called permeability transition pore (PTP), which accompanies a loss of transmembrane potential (⌬), a release of Ca 2ϩ and other cations, and mitochondrial swelling, causing ATP synthesis to stop in mitochondria (2-6). The PTP is thought to have a role in cellular Ca 2ϩ homeostasis, in import of mitochondrial protein, in thermal regulation in mammalian mitochondria, and as a common mediator of cell death (7,8).We focused on the role of adenine nucleotides in the modulation of MPT. The sensitivity of MPT to [Ca 2ϩ ] is greatly increased by ATP depletion (9). Matrix ADP is an important modulator of PTP opening and acts by decreasing the sensitivity of the calcium trigger site to Ca 2ϩ (10). The opening of PTP may be caused by well known membrane constituents, including the adenine nucleotide translocator (ANT), porin molecules, and the complex forming the peripheral benzodiazepine receptor (11,12). Two ADP binding sites may exist, one with a high affinity with the ANT that is blocked by the inhibitor atractyloside and the other site with a lower affinity (5, 13). These findings suggest that adenine nucleotides may be negative regulators of MPT.Mitochondrial damage seems to occur ...

β3 Adrenergic Stimulation Restores Nitric Oxide/Redox Balance and Enhances Endothelial Function in Hyperglycemia

“…Alloxan, the second most commonly used diabetogenic agent, increased β 1 pump subunit glutathionylation both in vitro and in vivo independently of diabetogenesis. These effects are consistent with ROS generation and thiol oxidizing properties of alloxan 32. In contrast to STZ and alloxan, S961 peptide had no effect on oxidative modification of β 1 pump subunit in vitro, suggesting that it did not have direct redox effects on the Na + ‐K + pump.…”

Induction of mitochondrial permeability transition by auranofin, a Gold(I)‐phosphine derivative

“…We have previously reported that an inhibitor of thioredoxin reductase such as 13‐ cis retinoic acid is also an inducer of permeability transition and apoptosis (Rigobello et al ., 1999). Similar considerations can be drawn for arsenicals, menadione and alloxan (Gunter & Pfeiffer, 1990; Zoratti & Szabò, 1995; Sakurai et al ., 2001; Arnèr et al ., 1999). More recently it was reported that ebselen, an antioxidant selenazol drug and glutathione peroxidase mimic (Sies, 1993), is able to induce swelling and calcium release (Gogvadze et al ., 2000) acting also as a direct substrate for thioredoxin reductase (Holmgren et al ., 2000).…”

“…We have previously demonstrated (Rigobello et al ., 1999) that 13‐ cis retinoic acid, an inhibitor of thioredoxin reductase (Schallreuter & Wood, 1989) is very effective in inducing mitochondrial permeability transition and release of cytochrome c . Similar effects on mitochondrial membranes permeability increase were observed with arsenite and arsenicals, heavy and transition metal cations, quinones, alloxan and sulfhydryl reagents (Gunter & Pfeiffer, 1990; Zoratti & Szabò, 1995; Sakurai et al ., 2001) that are also inhibitors or substrates of thioredoxin reductase. In the latter case they act as electron acceptors (Arnér et al ., 1999), therefore, diverting electrons from thioredoxin reductase that are no longer fed to its natural substrate, thioredoxin.…”

Mitochondrial Respiration Is Associated with Alloxan-Induced Mitochondrial Permeability Transition

“…The quinoidal compound alloxan has diabetogenic action. In previous reports we demonstrated that alloxan induced apoptotic death in INS-1 cells (a rat pancreatic β-cell line) and that the coincident mitochondrial damage is linked to apoptosis [8], and that alloxan induces MPT that requires mitochondrial energization, oxidation of protein thiols, and matrix ATP to promote energized uptake of Ca 2+ [9]. Several studies have demonstrated theinvolvement of mitochondrial events such as activation of mitochondrial-derived factors and the decrease in mitochondrial transmembrane potential in apoptosis [10] [11].…”

“…Several researchers have demonstrated that the diabetogenic action of alloxan is initiated by the generation of reactive oxygen species (ROS) [9] [14]- [16], althoughthe detailed molecular mechanism of alloxan-mediated cytotoxicity is not yet clearly understood. The initial step for ROS generation is mostly mediated by the mitochondrial electron transfer system [17].…”