The effect of permeability transition pore opening on reactive oxygen species production in rat brain mitochondria

The influence of mitochondrial permeability transition pore (MPTP) opening on reactive oxygen species (ROS) production in the rat brain mitochondria was studied. It was shown that ROS production is regulated differently by the rate of oxygen consumption and membrane potential, dependent on steady-state or non-equilibrium conditions. Under steady-state conditions, at constant rate of Ca2+-cycling and oxygen consumption, ROS production is potential-dependent and decreases with the inhibition of respiration and mitochondrial depolarization. The constant rate of ROS release is in accord with proportional dependence of the rate of ROS formation on that of oxygen consumption. On the contrary, transition to non-equilibrium state, due to the release of cytochrome c from mitochondria and progressive respiration inhibition, results in the loss of proportionality in the rate of ROS production on the rate of respiration and an exponential rise of ROS production with time, independent of membrane potential. Independent of steady-state or non-equilibrium conditions, the rate of ROS formation is controlled by the rate of potential-dependent uptake of Ca2+ which is the rate-limiting step in ROS production. It was shown that MPTP opening differently regulates ROS production, dependent on Ca2+ concentration. At low calcium MPTP opening results in the decrease in ROS production because of partial mitochondrial depolarization, in spite of sustained increase in oxygen consumption rate by a cyclosporine A-sensitive component due to simultaneous work of Ca2+-uniporter and MPTP as Ca2+-influx and efflux pathways. The effect of MPTP opening at low Ca2+ concentrations is similar to that of Ca2+-ionophore, A-23187. At high calcium MPTP opening results in the increase of ROS release due to the rapid transition to non-equilibrium state because of cytochrome c loss and progressive gating of electron flow in respiratory chain. Thus, under physiological conditions MPTP opening at low intracellular calcium could attenuate oxidative damage and the impairment of neuronal functions by diminishing ROS formation in mitochondria.     

Copper deficiency inhibits Ca2+-induced swelling in rat cardiac mitochondria

Cu deficiency disrupts the architecture of mitochondria, impairs respiration, and inhibits the activity of cytochrome c oxidase - the terminal, Cu-dependent respiratory complex (Complex IV) of the electron transport chain. This suggests that perturbations in the respiratory chain may contribute to the changes in mitochondrial structure caused by Cu deficiency. This study investigates the effect of Cu deficiency on Ca2+-induced mitochondrial swelling as it relates to changes in respiratory complex activities in cardiac mitochondria of rats. Male weanling rats were fed diets containing either no added Cu (Cu0), 1.5 mg Cu/kg (Cu1.5), 3 mg Cu/kg (Cu3) or 6 mg Cu/kg (Cu6). The rate of Ca2+-induced mitochondrial swelling in the presence of succinate and oligomycin was reduced, and the time to reach maximal swelling was increased only in the rats consuming Cu0 diet. Cytochrome c oxidase activity was reduced 60% and 30% in rats fed Cu0 and Cu1.5, respectively, while NADH:cytochrome c reductase (Complex I+ComplexIII) activity was reduced 30% in rats consuming both Cu0 and Cu1.5. Mitochondrial swelling is representative of mitochondrial permeability transition pore (MPTP) formation and the results suggest that Ca2+-induced MPTP formation occurs in cardiac mitochondria of Cu-deficient rats only when cytochrome c oxidase activity falls below 30% of normal. Decreased respiratory complex activities caused by severe Cu deficiency may inhibit MPTP formation by increasing matrix ADP concentration or promoting oxidative modifications that reduce the sensitivity of the calcium trigger for MPTP formation.     

Inhibition of Bcl-2 Sensitizes Mitochondrial Permeability Transition Pore (MPTP) Opening in Ischemia-Damaged Mitochondria

Background

Mitochondria are critical to cardiac injury during reperfusion as a result of damage sustained during ischemia, including the loss of bcl-2. We asked if bcl-2 depletion not only leads to selective permeation of the outer mitochondrial membrane (MOMP) favoring cytochrome c release and programmed cell death, but also favors opening of the mitochondrial permeability transition pore (MPTP). An increase in MPTP susceptibility would support a role for bcl-2 depletion mediated cell death in the calcium overload setting of early reperfusion via MPTP as well as later in reperfusion via MOMP as myocardial calcium content normalizes.

Methods

Calcium retention capacity (CRC) was used to reflect the sensitivity of the MPTP opening in isolated cardiac mitochondria. To study the relationship between bcl-2 inhibition and MPTP opening, mitochondria were incubated with a bcl-2 inhibitor (HA14-1) and CRC measured. The contribution of preserved bcl-2 content to MPTP opening following ischemia-reperfusion was explored using transgenic bcl-2 overexpressed mice.

Results

CRC was decreased in mitochondria following reperfusion compared to ischemia alone, indicating that reperfusion further sensitizes to MPTP opening. Incubation of ischemia-damaged mitochondria with increasing HA14-1concentrations increased calcium-stimulated MPTP opening, supporting that functional inhibition of bcl-2 during simulated reperfusion favors MPTP opening. Moreover, HA14-1 sensitivity was increased by ischemia compared to non-ischemic controls. Overexpression of bcl-2 attenuated MPTP opening in following ischemia-reperfusion. HA14-1 inhibition also increased the permeability of the outer membrane in the absence of exogenous calcium, indicating that bcl-2 inhibition favors MOMP when calcium is low.

Conclusions

The depletion and functional inhibition of bcl-2 contributes to cardiac injury by increasing susceptibility to MPTP opening in high calcium environments and MOMP in the absence of calcium overload. Thus, ischemia-damaged mitochondria with decreased bcl-2 content are susceptible to MPTP opening in early reperfusion and MOMP later in reperfusion when cytosolic calcium has normalized.     

Inhibition of the mitochondrial permeability transition by creatine kinase substrates. Requirement for microcompartmentation

Mitochondria from transgenic mice, expressing enzymatically active mitochondrial creatine kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of creatine kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by creatine or cyclocreatine but not by beta-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extramitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of creatine. If mitochondrial creatine kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by creatine or cyclocreatine was no longer seen. Likewise, when creatine kinase was added externally to liver mitochondria from wild-type mice that do not express mitochondrial creatine kinase in liver, no protective effect on pore opening by creatine and its analog was observed. All these findings indicate that mitochondrial creatine kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate mitochondrial permeability transition in the presence of creatine. These results are of relevance for the design of creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.     

Mitochondrial permeability transition relevance for apoptotic triggering in the post-ischemic heart

Dysfunction of mitochondrial calcium homeostasis transforms this cation from a key regulator of mitochondrial function, into a death effector during post-ischemic reperfusion. High intramitochondrial calcium and prevailing cellular conditions favor the opening of the mitochondrial permeability transition pore (mPTP), that induces mitochondrial swelling and provides a mechanism for cytochrome c release, a hallmark signal protein of the mitochondrial apoptosis pathway; indeed, a second mechanism induced by pro-apoptotic BAX protein, could account for cytochrome c leak in the post-ischemic heart. The present study was undertaken to determine which one of these mechanisms triggers the mitochondrial apoptosis pathway in the reperfused heart. To accomplish this goal we prevented the opening of the mPTP in such hearts, by diminishing calcium overload with Ru360, a specific mitochondrial calcium uniporter inhibitor. We found that mPTP opening in reperfused hearts increased along with reperfusion time and concurs with cytochrome c release from mitochondria. Maximal cytochrome c release correlated with mitochondrial dysfunction and complete NAD+ deletion. Fully inserted BAX was detected early after reperfusion and remained unchanged during the evaluated reperfusion times. Remarkably, heart perfusion with Ru360, inhibited mPTP opening and BAX docking into the mitochondrial membranes, suggesting a mPTP upstream role on BAX migration/insertion.     

In vivo hyperoxic preconditioning protects against rat-heart ischemia/reperfusion injury by inhibiting mitochondrial permeability transition pore opening and cytochrome c release

In vivo hyperoxic preconditioning (PC) has been shown to protect against ischemia/reperfusion (I/R) myocardial damage. Mitochondrial permeability transition pore (MPTP) opening is an important event in cardiomyocyte cell death occurring during I/R and therefore a possible target for cardioprotection. We tested the hypothesis that in vivo hyperoxic PC, obtained by mechanical ventilation of animals, could protect heart against I/R injury by inhibiting MPTP opening and cytochrome c release from mitochondria. Mechanically ventilated rats were first exposed to a short period of hyperoxia and isolated hearts were subsequently subjected to I/R in a Langendorff apparatus. Hyperoxic PC significantly improved the functional recovery of hearts on reperfusion, reduced the infarct size, and decreased necrotic damage as shown by the reduced release of lactate dehydrogenase. Mitochondria from hyperoxic PC hearts were less sensitive than mitochondria from reperfused heart to MPTP opening. In addition, hyperoxic PC prevented mitochondrial NAD(+) depletion, an indicator of MPTP opening, and cytochrome c release as well as cardiolipin oxidation/depletion associated with I/R. Together, these results demonstrate that hyperoxic PC protects against heart I/R injury by inhibiting MPTP opening and cytochrome c release. Thus, in vivo hyperoxic PC may represent a useful strategy for the treatment of cardiac I/R injury and could have potential applications in clinical practice.     

Mitochondria and cell death

Mitochondria play a central role in both apoptosis and necrosis through the opening of the mitochondrial permeability transition pore (MPTP). This is thought to be formed through a Ca(2+)-triggered conformational change of the adenine nucleotide translocase (ANT) bound to matrix cyclophilin-D and we have now demonstrated this directly by reconstitution of the pure components. Opening of the MPTP causes swelling and uncoupling of mitochondria which, unrestrained, leads to necrosis. In ischaemia/reperfusion injury of the heart we have shown MPTP opening directly. Recovery of hearts correlates with subsequent closure, and agents that prevent opening or enhance closure protect from injury. Transient MPTP opening may also be involved in apoptosis by initially causing swelling and rupture of the outer membrane to release cytochrome c (cyt c), which then activates the caspase cascade and sets apoptosis in motion. Subsequent MPTP closure allows ATP levels to be maintained, ensuring that cell death remains apoptotic rather than necrotic. Apoptosis in the hippocampus that occurs after a hypoglycaemic or ischaemic insult is triggered by this means. Other apoptotic stimuli such as cytokines or removal of growth factors also involve mitochondrial cyt c release, but here there is controversy over whether the MPTP is involved. In many cases cyt c release is seen without any mitochondrial depolarization, suggesting that the MPTP does not open. Recent data of our own and others have revealed a specific outer-membrane cyt c-release pathway involving porin that does not release other intermembrane proteins such as adenylate kinase. This is opened by pro-apoptotic members of the Bcl-2 family such as BAX and prevented by anti-apoptotic members such as Bcl-X(L). Our own data suggest that this pathway may interact directly with the ANT in the inner membrane at contact sites.     

BH3 death domain peptide induces cell type-selective mitochondrial outer membrane permeability

The BH3 domain is essential for the release of cytochrome c from mitochondria by pro-apoptotic Bcl-2 family proteins during apoptosis. This study tested the hypothesis that a Bax peptide that includes the BH3 domain can permeabilize the mitochondrial outer membrane and release cytochrome c in the absence of a permeability transition at the mitochondrial inner membrane. BH3 peptide (0.1-60 microm) released cytochrome c from mitochondria in the presence of physiological concentrations of ions in a cell type-selective manner, whereas a BH3 peptide with a single amino acid substitution was ineffective. The release of cytochrome c by BH3 peptide correlated with the presence of endogenous Bax at the mitochondria and its integral membrane insertion. Cytochrome c release was accompanied by adenylate kinase release, was not associated with mitochondrial swelling or substantial loss of electrical potential across the inner membrane, and was unaffected by inhibitors of the permeability transition pore. Cytochrome c release was, however, inhibited by Bcl-2. Although energy-coupled respiration was inhibited after the release of cytochrome c, mitochondria maintained membrane potential in the presence of ATP due to the reversal of the ATP synthase. Overall, results support the hypothesis that BH3 peptide releases cytochrome c by a Bax-dependent process that is independent of the mitochondrial permeability transition pore but regulated by Bcl-2.     

线粒体渗透性转变孔结构和功能的研究进展

非正常生理浓度的Ca^2 和氧化应激等刺激线粒体渗透性转变孔(mitochondria permeability transition pore,MPTP)开放,使线粒体形态功能发生改变,被释放的细胞色素c和凋亡诱导因子(apoptosisinducing factor,AIF)等参与到caspase信号通路中,诱导细胞发生凋亡。本文在MPTP的主要组成成分、两种不同的结构功能模型、抑制剂对MPTP的抑制机制和缺血,再灌注及缺血预适应对MPTP开放的影响等方面的研究进展作一综述。     

Silencing of cardiac mitochondrial NHE1 prevents mitochondrial permeability transition pore opening

Inhibition of Na(+)/H(+) exchanger 1 (NHE1) reduces cardiac ischemia-reperfusion (I/R) injury and also cardiac hypertrophy and failure. Although the mechanisms underlying these NHE1-mediated effects suggest delay of mitochondrial permeability transition pore (MPTP) opening, and reduction of mitochondrial-derived superoxide production, the possibility of NHE1 blockade targeting mitochondria has been incompletely explored. A short-hairpin RNA sequence mediating specific knock down of NHE1 expression was incorporated into a lentiviral vector (shRNA-NHE1) and transduced in the rat myocardium. NHE1 expression of mitochondrial lysates revealed that shRNA-NHE1 transductions reduced mitochondrial NHE1 (mNHE1) by ～60%, supporting the expression of NHE1 in mitochondria membranes. Electron microscopy studies corroborate the presence of NHE1 in heart mitochondria. Immunostaining of rat cardiomyocytes also suggests colocalization of NHE1 with the mitochondrial marker cytochrome c oxidase. To examine the functional role of mNHE1, mitochondrial suspensions were exposed to increasing concentrations of CaCl(2) to induce MPTP opening and consequently mitochondrial swelling. shRNA-NHE1 transduction reduced CaCl(2)-induced mitochondrial swelling by 64 ± 4%. Whereas the NHE1 inhibitor HOE-642 (10 μM) decreased mitochondrial Ca(2+)-induced swelling in rats transduced with nonsilencing RNAi (37 ± 6%), no additional HOE-642 effects were detected in mitochondria from rats transduced with shRNA-NHE1. We have characterized the expression and function of NHE1 in rat heart mitochondria. Because mitochondria from rats injected with shRNA-NHE1 present a high threshold for MPTP formation, the beneficial effects of NHE1 inhibition in I/R resulting from mitochondrial targeting should be considered.     

Mersalyl prevents the Tl+-induced permeability transition pore opening in the inner membrane of Ca2+-loaded rat liver mitochondria

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO₃ and KNO₃ resulted in the Tl⁺-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca²⁺ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl⁺-induced MPTP opening in the inner membrane of Ca²⁺-loaded rat liver mitochondria.     

The role of mitochondrial permeability transition pore in transmembrane Ca2+-exchange in mitochondria

Ca2+-uptake accompanied with mitochondrial permeability transition pore (MPTP) opening is studied in rat liver mitochondria. In conditions of MPTP opening, as well as in conditions of MPTP blockage by cyclosporine A (CsA), Ca2+-uptake in mitochondria is counterbalanced by proton efflux into incubation medium. Independent of MPTP opening, observed stoichiometry of this exchange is 1Ca2+ : 1H+. MPTP opening dramatically decreases Ca2+-uptake in mitochondria: from approximately 400 nmol/mg protein in the presence of CsA to approximately 80-100 nmol/mg protein due to the increased mitochondrial membrane permeability. In the absence of CsA Ca2+-uptake is accompanied by the insensitive to Ca2+-uniporter blocker, ruthenium red (RR), release of Ca2+ from mitochondria which corresponds to as well RR-insensitive, but sensitive to CsA uptake of H+ into mitochondrial matrix. This calcium-proton exchange resulting from MPTP opening is observed only when Ca2+ uptake into matrix exceeds some basal level. The data are consistent with an assumption that, contrary to Ca2+-uniporter, MPTP has its own proton conductance. MPTP opening provides exchange of Ca2+ between mitochondria and medium which is coupled to the counterflow of protons into matrix space. Obtained data elucidate the physiological role of MPTP as regulatory mechanism for control of Ca2+-uptake level and intramitochondrial pH.     

Role of alpha-tocopherol in the regulation of mitochondrial permeability transition

We previously showed that Ca2+-induced cyclosporin A-sensitive membrane permeability transition (MPT) of mitochondria occurred with concomitant generation of reactive oxygen species (ROS) and release of cytochrome c (Free Rad. Res.38, 29-35, 2004). To elucidate the role of alpha-tocopherol in MPT, we investigated the effect of alpha-tocopherol on mitochondrial ROS generation, swelling and cytochrome c release induced by Ca2+ or hydroxyl radicals. Biochemical analysis revealed that alpha-tocopherol suppressed Ca2+-induced ROS generation and oxidation of critical thiol groups of mitochondrial adenine nucleotide translocase (ANT) but not swelling and cytochrome c release. Hydroxyl radicals also induced cyclosporin A-sensitive MPT of mitochondria. alpha-Tocopherol suppressed the hydroxyl radical-induced lipid peroxidation, swelling and cytochrome c release from mitochondria. These results indicate that alpha-tocopherol inhibits ROS generation, ANT oxidation, lipid peroxidation and the opening of MPT, thereby playing important roles in the prevention of oxidative cell death.     

Neonatal cardiac mitochondria and ischemia/reperfusion injury

Postnatal maturation of the heart is characterized by decreasing tolerance to ischemia/reperfusion (I/R) injury associated with significant changes in mitochondrial function. The aim of this study is to test the hypothesis that the role of the mitochondrial membrane permeability transition pore (MPTP) in the I/R injury differs in the neonatal and in the adult heart. For this purpose, the effect of blockade of MPTP on the degree of I/R injury and the sensitivity of MPTP to swelling-inducing agents was compared in hearts from neonatal (7 days old) and adult (90 days old) Wistar rats. It was found that the release of NAD⁺ from the perfused heart induced by I/R can be prevented by sanglifehrin A (SfA) only in the adult myocardium; SfA had no protective effect in the neonatal heart. Furthermore, the extent of Ca-induced swelling of mitochondria from neonatal rats was significantly lower than that from the adult animals; mitochondria from neonatal rats were more resistant at higher concentrations of calcium. In addition, not only the extent but also the rate of calcium-induced swelling was about twice higher in adult than in neonatal mitochondria. The results support the idea that lower sensitivity of the neonatal MPTP to opening may be involved in the mechanism of the higher tolerance of the neonatal heart to I/R injury.     

Ca(2+)-induced high amplitude swelling and cytochrome c release from wheat (Triticum aestivum L.) mitochondria under anoxic stress

Under stress conditions, mitochondria sense metabolic changes, e.g. in pH, cytoplasmic Ca(2+), energy status, and reactive oxygen species (ROS), and respond by induction of the permeability transition pore (PTP) and by releasing cytochrome c, thus initiating the programmed cell death (PCD) cascade in animal cells. In plant cells, the presence of all the components of the cascade has not yet been shown. In wheat (Triticum aestivum L.) root mitochondria, the onset of anoxia caused rapid dissipation of the inner membrane potential, initial shrinkage of the mitochondrial matrix and the release of previously accumulated Ca(2+). Ca(2+) uptake by mitochondria was dependent on the presence of inorganic phosphate. Treatment of mitochondria with high micromolar and millimolar Ca(2+) (but not Mg(2+)) concentrations induced high amplitude swelling, indicative of PTP opening. Alterations in mitochondrial volume were confirmed by transmission electron microscopy. Mitochondrial swelling was not sensitive to cyclosporin A (CsA)-an inhibitor of mammalian PTP. The release of cytochrome c was monitored under lack of oxygen. Anoxia alone failed to induce cytochrome c release from mitochondria. Oxygen deprivation and Ca(2+) ions together caused cytochrome c release in a CsA-insensitive manner. This process correlated positively with Ca(2+) concentration and required Ca(2+) localization in the mitochondrial matrix. Functional characteristics of wheat root mitochondria, such as membrane potential, Ca(2+) transport, swelling, and cytochrome c release under lack of oxygen are discussed in relation to PCD.     

Ganglioside GD3 and its mimetics induce cytochrome c release from mitochondria

Ganglioside GD3 induced the release of cytochrome c from isolated rat liver mitochondria. This process was completely prevented by cyclosporin A and partially prevented by a cysteine protease inhibitor, n-acetyl-leu-leu-norleucinal. Cyclosporin A is a potent inhibitor of the permeability transition pore, whereas n-acetyl-leu-leu-norleucinal has no effect on this pore. These results indicate that the release of cytochrome c from mitochondria requires both the opening of the permeability transition pore and a cysteine protease inhibitor-sensitive mechanism. Gangliosides GD1a, GD1b, GT1b, and GQ1b along with the synthetic GD3 mimetics TMS-42 and CI-22, which are glycerophospholipids carrying a disialo residue, also induced cytochrome c release. In contrast, gangliosides GM1, GM2, and GM3 did not induce cytochrome c release. These results indicate that two sialo residues must play an important role in the induction of cytochrome c release by gangliosides.     

Bcl-2 sensitive mitochondrial potassium accumulation and swelling in apoptosis

During etoposide-induced apoptosis in HL-60 cells, cytochrome c release was associated with mitochondrial swelling caused by increased mitochondrial potassium uptake. The mitochondrial permeability transition was also observed; however, it was not the primary cause of mitochondrial swelling. Potassium uptake and swelling of mitochondria were blocked by bcl-2 overexpression. As a result, cytochrome c release was reduced, and apoptosis delayed. Residual cytochrome c release in the absence of swelling in bcl-2 expressing cells could be due to observed Bax translocation into mitochondria. This study suggests several novel aspects of apoptotic signaling: (1) potassium related swelling of mitochondria; (2) inhibition of mitochondrial potassium uptake by bcl-2; (3) co-existence within one system of multiple mechanisms of cytochrome c release: mitochondrial swelling and swelling-independent permeabilization of the outer mitochondrial membrane.     

Chenodeoxycholate induction of mitochondrial permeability transition pore is associated with increased membrane fluidity and cytochrome c release: protective role of carvedilol

Chenodeoxycholate (CDCA) is a primary bile acid mostly implicated in cholestatic liver injury. In this study, we have investigated the involvement of membrane fluidity and cytochrome c release in CDCA-induced mitochondrial permeability transition (MPT), and the preventive role of carvedilol. Treatment of calcium-loaded hepatic mitochondria with CDCA was found to cause osmotic swelling and release of cytochrome c, associated with an increase in membrane fluidity, in both protein and lipid regions. Carvedilol and cyclosporine A (CyA) reduced both cytochrome c release and alterations in membrane fluidity induced by CDCA. The hydroxylated metabolite of carvedilol, BM-910228, had no effect. Thus, modulation of membrane fluidity, plays an important role in MPT pore opening promoted by CDCA. As a result, we have delineated a pathway for the preventive role of carvedilol in mitochondrial dysfunction induced by CDCA.     

A study on permeability transition pore opening and cytochrome c release from mitochondria, induced by caspase-3 in vitro.

We recently described that there is a feedback amplification of cytochrome c release from mitochondria by caspases. Here we investigated how caspases impact on mitochondria to induce cytochrome c release and found that recombinant caspase-3 induced opening of permeability transition pore and reduction of membrane potential in vitro. These events were inhibited by Bcl-xL, cyclosporin A and z-VAD.fmk. Moreover, caspase-3 stimulated the rate of mitochondrial state 4 respiration, superoxide production and NAD(P)H oxidation in a Bcl-xL- and cyclosporin A-inhibitable manner. These results suggest that caspase-3 induces cytochrome c release by inducing permeability transition pore opening which is associated with changes in mitochondrial respiration and redox potential.     

Absence of Ca2+-induced mitochondrial permeability transition but presence of bongkrekate-sensitive nucleotide exchange in C. crangon and P. serratus

Mitochondria from the embryos of brine shrimp (Artemia franciscana) do not undergo Ca(2+)-induced permeability transition in the presence of a profound Ca(2+) uptake capacity. Furthermore, this crustacean is the only organism known to exhibit bongkrekate-insensitive mitochondrial adenine nucleotide exchange, prompting the conjecture that refractoriness to bongkrekate and absence of Ca(2+)-induced permeability transition are somehow related phenomena. Here we report that mitochondria isolated from two other crustaceans, brown shrimp (Crangon crangon) and common prawn (Palaemon serratus) exhibited bongkrekate-sensitive mitochondrial adenine nucleotide transport, but lacked a Ca(2+)-induced permeability transition. Ca(2+) uptake capacity was robust in the absence of adenine nucleotides in both crustaceans, unaffected by either bongkrekate or cyclosporin A. Transmission electron microscopy images of Ca(2+)-loaded mitochondria showed needle-like formations of electron-dense material strikingly similar to those observed in mitochondria from the hepatopancreas of blue crab (Callinectes sapidus) and the embryos of Artemia franciscana. Alignment analysis of the partial coding sequences of the adenine nucleotide translocase (ANT) expressed in Crangon crangon and Palaemon serratus versus the complete sequence expressed in Artemia franciscana reappraised the possibility of the 208-214 amino acid region for conferring sensitivity to bongkrekate. However, our findings suggest that the ability to undergo Ca(2+)-induced mitochondrial permeability transition and the sensitivity of adenine nucleotide translocase to bongkrekate are not necessarily related phenomena.