Ca(2+) efflux in mitochondria from the yeast Endomyces magnusii.

Calcium release pathways in Ca(2+)-preloaded mitochondria from the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca(2+) was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca(2+) release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca(2+) and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca(2+) efflux set in after uptake of approximately 150 nmol of Ca(2+)/mg of protein. The rate of this efflux was proportional to the Ca(2+) load and insensitive to aeration, protonophorous uncouplers, and Na(+) ions. Ca(2+) efflux was inhibited by La(3+), Mn(2+), Mg(2+), tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca(2+) efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca(2+) efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na(+)-independent pathway, in many ways similar to the one in mammalian mitochondria.     

Gliotoxin induces Mg2+ efflux from intact brain mitochondria

Gliotoxin (GT) is a hydrophobic fungal metabolite of the epipolythiodioxopiperazine group which reacts with membrane thiols. When added to a suspension of energized brain mitochondria, it induces matrix swelling of low amplitude, collapse of membrane potential (DeltaPsi), and efflux of endogenous cations such as Ca2+ and Mg2+, typical events of mitochondrial permeability transition (MPT) induction. These effects are due to opening of the membrane transition pore. The addition of cyclosporin A (CsA) or ADP slightly reduces membrane potential collapse, matrix swelling and Ca2+ efflux; Mg2+ efflux is not affected at all. The presence of exogenous Mg2+ or spermine completely preserve mitochondria against DeltaPsi collapse, matrix swelling and Ca2+ release. Instead, Mg2+ efflux is only slightly affected by spermine. Our results demonstrate that, besides inducing MPT, gliotoxin activates a specific Mg2+ efflux system from brain mitochondria.     

Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A

Ca(2+) plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca(2+) is controlled primarily by the mitochondrial Ca(2+) uniporter and the mitochondrial Na(+)/Ca(2+) exchanger, influencing NADH production through Ca(2+)-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca(2+)-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca(2+) release. Here we selectively measure Ca(2+) influx rate through the mitochondrial Ca(2+) uniporter and Ca(2+) efflux rates through Na(+)-dependent and Na(+)-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na(+)/Ca(2+) exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ(m) loss, Ca(2+) release, NADH oxidation, swelling) of high extramitochondrial Ca(2+) additions, allowing mitochondria to tolerate total mitochondrial Ca(2+) loads of >400nmol/mg protein. For Ca(2+) pulses up to 15μM, Na(+)-independent Ca(2+) efflux through the permeability transition pore accounted for ~5% of the total Ca(2+) efflux rate compared to that mediated by the mitochondrial Na(+)/Ca(2+) exchanger (in 5mM Na(+)). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na(+)/Ca(2+) exchanger-mediated Ca(2+) efflux at higher concentrations (IC(50)=2μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~40% at 10μM cyclosporine A, while having no effect on the mitochondrial Ca(2+) uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca(2+) load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Ca2+-release pathways from mitochondria of the yeast Endomyces magnusii

Ca2+-release pathways from Ca2+-preloaded mitochondria of the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca2+ was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Intensive aeration of the mitochondrial suspension rapidly inhibited the efflux of Ca2+ and induced its reuptake. The Ca2+ release was not affected by cyclosporin A, an inhibitor of the nonselective permeability transition of mammalian mitochondria. With acetate as the permeant anion, a spontaneous net Ca2+ efflux began after uptake of about 75% of the added cation. The rate of this efflux was insensitive to cyclosporin A, aeration, and Na+ and was proportional to the Ca2+ load. The Ca2+ release was inhibited by La3+, Mn2+, Mg2+, TPP+, and nigericin (in the presence of KCl) and activated by spermine and hypotonicity. We conclude that Ca2+ efflux from preloaded E. magnusii mitochondria is very similar to the Na+-independent specific pathway for Ca2+ release operative in mitochondria from nonexcitable mammalian tissues.     

The role of Fe2+-induced lipid peroxidation in the initiation of the mitochondrial permeability transition

Iron and iron complexes stimulate lipid peroxidation and formation of malondialdehyde (MDA). We have studied the effects of Fe2+ and ascorbate on mitochondrial permeability transition induced by phosphate and Ca2+. Iron is necessary for detectable MDA formation, but only Ca2+ and phosphate are necessary for the induction of membrane potential loss (Deltapsi) and Ca2+ release. Keeping the iron at a constant concentration and varying the Ca2+ level changed the mitochondrial Ca2+ retention times, but not the amount of MDA formation. The antioxidant butylated hydroxytoluene at low concentrations prevented MDA formation, but not mitochondrial Ca2+ release. Preincubation of mitochondria with Fe2+ decreased Ca2+ retention time in a concentration-dependent manner and facilitated Ca2+-stimulated MDA accumulation. Thus, Ca2+ phosphate-induced mitochondrial permeability transition (MPT) can be separated mechanistically from MDA accumulation. Lipid peroxidation products do not appear to participate in the initial phase of the permeability transition, but sensitize mitochondria toward MPT.     

Mitochondrial iPLA2 activity modulates the release of cytochrome c from mitochondria and influences the permeability transition

The mitochondrial Ca(2+)-independent phospholipase A(2) is activated during energy-dependent Ca(2+) accumulation under conditions where there is a sustained depression of the membrane potential. This activation is not dependent on induction of the mitochondrial permeability transition. Bromoenol lactone, which inhibits the phospholipase, is effective as an inhibitor of the transition, and this action can be overcome by low levels of exogenous free fatty acids. Apparently, activation of the Ca(2+)-independent phospholipase is a factor in the mechanisms by which depolarization and Ca(2+) accumulation promote opening of the permeability transition pore. Sustained activity of the Ca(2+)-independent phospholipase A(2) promotes rupture of the outer mitochondrial membrane and spontaneous release of cytochrome c on a time scale similar to that of apoptosis occurring in cells. However, more swelling of the matrix space must occur to provoke release of a given cytochrome c fraction when the enzyme is active, compared with when it is inhibited. Through its effects on the permeability transition and release of intermembrane space proteins, the mitochondrial Ca(2+)-independent phospholipase A(2) may be an important factor governing cell death caused by necrosis or apoptosis.     

Calcium-Mediated Mitochondrial Permeability Transition Involved in Hydrogen Peroxide-Induced Apoptosis in Tobacco Protoplasts

In the present study, we focused on whether Intracellular free Ca^2＋ （[Ca^2＋],） regulates the formation of mltochondrlal permeability transition pore （MPTP） In H2O2-induced apoptosis In tobacco protoplasts. It was shown that the decrease In mltochondrlal membrane potential （△ψm） preceded the appearance of H2O2-Induced apoptosls; pretreatment with the specific MPTP Inhibitor cyclosporine A, which also Inhibits Ca^2＋ cycling by the mitochondria, effectively retarded apoptosls and the decrease In △ψm. Apoptosls and decreased △ψm were exacerbated by CaCl2, whereas the plasma membrane voltage-dependent Ca^2＋ channel blocker lanthanum chloride （LaCl3） attentuated these responses. Chelation of extracellular Ca^2＋ with EGTA almost totally Inhibited apoptosls and the decrease In △ψmInduced by H2O2. The time-course of changes In [Ca^2＋]l In apoptosls was detected using the Ca^2＋ probe Fiuo-3 AM. These studies showed that [Ca^2＋]1 was Increased at the very early stage of H2O2-Induced apoptosls. The EGTA evidently Inhibited the Increase In [Ca^2＋]1 Induced by H=O=, whereas It was only partially Inhibited by LaCl3. The results suggest that H2O2 may elevate cytoplasmic free Ca^2＋ concentrations In tobacco protoplasts, which mainly results from the entry of extracellular Ca^2＋, to regulate mltochondrlal permeability transition. The signaling pathway of [Ca^2＋]1-medlated mltochondrlal permeability transition was associated with H2O2-Induced apoptosis In tobacco protoplaete.     

Spermine. A regulator of mitochondrial calcium cycling

Steady-state free Ca2+ concentrations have been measured with a Ca2+ electrode using suspensions of isolated rat liver mitochondria or saponin-treated hepatocytes. Mitochondria, when incubated in the presence of Mg2+ and MgATP2-, maintain a steady-state pCa2+ (-log [Ca2+]) of approximately 6.1 (0.8 microM). Addition of spermine lowered this value to a pCa2+ of 6.6 (0.25 microM). Spermine was the most effective polyamine, giving half-maximal effects at 170 microM and maximal effects at 400 microM. With saponin-permeabilized hepatocytes, spermine addition similarly showed that the mitochondria buffered the steady-state medium-free Ca2+ at a level approximating the cytosolic free Ca2+ concentration of intact hepatocytes. The initial rate of Ca2+ uptake by the mitochondrial Ca2+ uniporter was investigated using Ca2+-depleted mitochondria incubated in the presence of succinate and 0.3 mM free Mg2+. Under control conditions, Ca2+ uptake was not observed at free Ca2+ concentrations below 0.5 microM. Spermine (350 microM) increased the rate of Ca2+ uptake at all Ca2+ concentrations below 4.5 microM, but at higher Ca2+ concentrations, it was inhibitory. Spermine also affected mitochondrial Ca2+ efflux by decreasing the apparent Km from 16 to 3.8 nmol of Ca2+/mg of mitochondrial protein with no change of Vmax. Experiments with 45Ca2+ confirmed that spermine increased mitochondrial Ca2+ cycling at 0.2 microM free Ca2+. Hepatic spermine contents are reported to be about 1 mumol/g, wet weight, suggesting that this polyamine may have an important physiological role in intracellular calcium homeostasis.     

Zinc as an inducer of the membrane permeability transition in rat liver mitochondria

It is shown that 2-10 microM Zn2+ induces swelling of rat liver mitochondria incubated in a buffered sucrose medium either with valinomycin or with FCCP, Ca2+, ionophore A23187, oligomycin, and nigericin. This swelling was associated with the release of GSH from mitochondria. Both processes were sensitive to known inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A, and Mg2+. Mitochondrial swelling induced by Zn2+ was also inhibited by rotenone, antymycin A, N-ethylmaleimide, butylhydroxytoluene, and spermine, whereas it was stimulated by tert-butyl hydroperoxide, diamide, and monobromobimane. It did not require the addition of phosphate. The same sensitivity to pH of the mitochondrial swelling induced by Zn2+ and by phenylarsine oxide suggests the same site of the interaction, namely, thiol groups. The ability of Zn2+ to induce mitochondrial swelling gradually decreased along with its increasing concentration above 10 microM. It is concluded that micromolar Zn2+ induces the MPT presumably by the interaction with cysteinyl residues. This process is independent of the mitochondrial membrane potential.     

Interactions between spermine and Mg2+ on mitochondrial Ca2+ transport

The effects of the polyamine spermine on the regulation of Ca2+ transport by subcellular organelles from rat liver, heart, and brain were investigated using ion-sensitive minielectrodes and a 45Ca2+ tracer method. Spermine stimulated Ca2+ uptake by mitochondria but not by microsomes. In the presence of spermine, isolated mitochondria could maintain a free extramitochondrial Ca2+ concentration of 0.3-0.2 microM. Stimulation of the initial rates of Ca2+ uptake and 45Ca2+ cycling of mitochondria by spermine shows that this was accomplished through a decrease of the apparent Km for Ca2+ uptake by the Ca2+ uniporter. The half maximally effective concentration of spermine (50 microM) was in the range of physiological concentrations of this polyamine in the cell. Spermidine was five times less effective. Putrescine was ineffective. The stimulation of mitochondrial Ca2+ uptake by spermine was inhibited by Mg2+ in a concentration-dependent manner. However, the diminished contribution of the mitochondria to the regulation of the free extraorganellar Ca2+ concentration could mostly be compensated for by microsomal Ca2+ uptake. Spermine also reversed ruthenium red-induced Ca2+ efflux from mitochondria. It is concluded that spermine is an activator of the mitochondrial Ca2+ uniporter and Mg2+ an antagonist. By this mechanism, the polyamines can confer to the mitochondria an important role in the regulation of the free cytoplasmic Ca2+ concentration in the cell and of the free Ca2+ concentration in the mitochondrial matrix.     

Bax does not directly participate in the Ca(2+)-induced permeability transition of isolated mitochondria

The mitochondrial permeability transition pore and Bax have both been proposed to be involved in the release of pro-apoptotic factors from mitochondria in the "intrinsic" pathway of apoptosis. The permeability transition pore is widely thought to be a supramolecular complex including or interacting with Bax. Given the relevance of the permeability transition in vivo, we have verified whether Bax influences the formation and/or the properties of the Ca(2+)/P(i)-induced permeability transition by using mitochondriaisolated from isogenic human colon cancer bax(+/-) and bax(-/-) HCT116 cell lines. We used mitochondria isolated from both types of cells and from Bax(+) cells exposed to apoptotic stimuli, as well as Bax-less mitochondria into which exogenous Bax had been incorporated. All exhibited the same behavior and pharmacological profile in swelling and Ca(2+)-retention experiments. Mitochondria from a bax(-)/bak(-) cell line also underwent an analogous Ca(2+)/P(i)-inducible swelling. This similarity indicates that Bax hasno major role in regulating the Ca(2+)-induced mitochondrial permeability transition.     

Prooxidants open both the mitochondrial permeability transition pore and a low-conductance channel in the inner mitochondrial membrane

Mitochondria can be induced by a variety of agents/conditions to undergo a permeability transition (MPT), which nonselectively increases the permeability of the inner membrane (i.m.) to small (<1500 Da) solutes. Prooxidants are generally considered to trigger the MPT, but some investigators suggest instead that prooxidants open a Ca(2+)-selective channel in the inner mitochondrial membrane and that the opening of this channel, when coupled with Ca(2+) cycling mediated by the Ca(2+) uniporter, leads ultimately to the observed increase in mitochondrial permeability [see, e.g., Schlegel et al. (1992) Biochem. J. 285, 65]. S. A. Novgorodov and T. I. Gudz [J. Bioenerg. Biomembr. (1996) 28, 139] propose that the i.m. contains a pore that, upon exposure to prooxidants, can open to two states, one of which conducts only H(+) and one of which is the classic MPT pore. Given the current interest in increased mitochondrial permeability as a factor in apoptotic cell death, it is important to determine whether i.m. permeability is regulated in one or multiple ways and, in the latter event, to characterize each regulatory mechanism in detail. This study examined the effects of the prooxidants diamide and t-butylhydroperoxide (t-BuOOH) on the permeability of isolated rat liver mitochondria. Under the experimental conditions used, t-BuOOH induced mitochondrial swelling only in the presence of exogenous Ca(2+) (>2 microM), whereas diamide was effective in its absence. In the absence of exogenous inorganic phosphate (P(i)), (1) both prooxidants caused a collapse of the membrane potential (DeltaPsi) that preceded the onset of mitochondrial swelling; (2) cyclosporin A eliminated the swelling induced by diamide and dramatically slowed that elicited by t-BuOOH, without altering prooxidant-induced depolarization; (3) collapse of DeltaPsi was associated with Ca(2+) efflux but not with efflux of glutathione; (4) neither Ca(2+) efflux nor DeltaPsi collapse was sensitive to ruthenium red; (5) collapse of DeltaPsi was accompanied by an increase in matrix pH; no stimulation of respiration was observed; (6) Sr(2+) was able to substitute for Ca(2+) in supporting t-BuOOH-induced i.m. depolarization, but not swelling; (7) in addition to being insensitive to CsA, the collapse of DeltaPsi was also resistant to trifluoperazine, spermine, and Mg(2+), all of which block the MPT; and (8) DeltaPsi was restored (and its collapse was inhibited) upon addition of dithiothreitol, ADP, ATP or EGTA. We suggest that these results indicate that prooxidants open two channels in the i.m.: the classic MPT and a low-conductance channel with clearly distinct properties. Opening of the low-conductance channel requires sulfhydryl group oxidation and the presence of a divalent cation; both Ca(2+) and Sr(2+) are effective. The channel permits the passage of cations, including Ca(2+), but not of protons. It is insensitive to inhibitors of the classic MPT.     

Changes in calcium-dependent membrane permeability properties in mitochondria of livers from arthritic rats

The involvement of the mitochondrial permeability transition pore (PTP) in the responses of mitochondria from adjuvant-induced arthritic rats to Ca(2+) addition was investigated. The respiratory activity, the Ca(2+)-induced osmotic swelling and the electrophoretic (45)Ca(2+) uptake were evaluated in the absence and in the presence of cyclosporin A (CsA), a well-known inhibitor of the mitochondrial PTP. The Ca(2+)-induced mitochondrial permeability transition (MPT) process occurred in mitochondria from arthritic rats even in the presence of a low Ca(2+) concentration. Whereas in the normal condition, the Ca(2+)-induced uncoupling of oxidative phosphorylation and osmotic swelling was observed in the presence of 10 or 20 microM Ca(2+) concentration, in the arthritic condition, these events occurred at 1.0 microM concentration. In addition, mitochondria from arthritic rats presented an impaired ability to accumulate (45)Ca(2+). All these effects were completely prevented by the administration of CsA. The results of the present study suggest that the higher sensitivity of mitochondria from arthritic rats to Ca(2+)-induced MPT may be an important factor in the pathogenesis of the arthritis disease.     

Effects of polyamines on mitochondrial Ca(2+) transport

Mammalian mitochondria are able to enhance Ca(2+) accumulation in the presence of polyamines by activating the saturable systems of Ca(2+) inward transport and buffering extramitochondrial Ca(2+) concentrations to levels similar to those in the cytosol of resting cells. This effect renders them responsive to regulate free Ca(2+) concentrations in the physioloical range. The mechanism involved is due to a rise in the affinity of the Ca(2+) transport system, induced by polyamines, most probably exhibiting allosteric behaviour. The regulatory site of this mechanism is the so-called S(1) binding site of polyamines, which operates in physiological conditions and is located in the energy well between the two peaks present in the energy profile of mitochondrial spermine transport. Spermine is bidirectionally transported across teh inner membrane by cycling, in which influx and efflux are driven by electrical and pH gradients, respectively. Most probably, polyamine affects the Ca(2+) transport system when it acts from the outside-that is, in the direction of its uniporter channel, in order to reach the S(1) site. Important physiological functions are related to activation of Ca(2+) transport systems by polyamines and their interactions with the S(1) site. These functions include a rise in the metabolic rate for energy supply and modulation of mitochondrial permeability transition induction, with consequent effects on the triggering of the apoptotic pathway.     

Calcium influx through receptor-operated channel induces mitochondria-triggered paraptotic cell death

We address the specific role of cytoplasmic Ca(2+) overload as a cell death trigger by expressing a receptor-operated specific Ca(2+) channel, vanilloid receptor subtype 1 (VR1), in Jurkat cells. Ca(2+) uptake through the VR1 channel, but not capacitative Ca(2+) influx stimulated by the muscarinic type 1 receptor, induced sustained intracellular [Ca(2+)] rises, exposure of phosphatidylserine, and cell death. Ca(2+) influx was necessary and sufficient to induce mitochondrial damage, as assessed by opening of the permeability transition pore and collapse of the mitochondrial membrane potential. Ca(2+)-induced cell death was inhibited by ruthenium red, protonophore carbonyl cyanide m-chlorophenylhydrazone, or cyclosporin A treatment, as well as by Bcl-2 expression, indicating that this process requires mitochondrial calcium uptake and permeability transition pore opening. Cell death occurred without caspase activation, oligonucleosomal/50-kilobase pair DNA cleavage, or release of cytochrome c or apoptosis inducer factor from mitochondria, but it required oxidative/nitrative stress. Thus, Ca(2+) influx triggers a distinct program of mitochondrial dysfunction leading to paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis.     

The oat mitochondrial permeability transition and its implication in victorin binding and induced cell death

The mitochondrion has emerged as a key regulator of apoptosis, a form of animal programmed cell death (PCD). The mitochondrial permeability transition (MPT), facilitated by a pore-mediated, rapid permeability increase in the inner membrane, has been implicated as an early and critical step of apoptosis. Victorin, the host-selective toxin produced by Cochliobolus victoriae, the causal agent of victoria blight of oats, has been demonstrated to bind to the mitochondrial P-protein and also induces a form of PCD. Previous results suggest that a MPT may facilitate victorin's access to the mitochondrial matrix and binding to the P-protein: (i) victorin-induced cell death displays features similar to apoptosis; (ii) in vivo, victorin binds to the mitochondrial P-protein only in toxin-sensitive genotypes whereas victorin binds equally well to P-protein isolated from toxin-sensitive and insensitive oats; (iii) isolated, untreated mitochondria are impermeable to victorin. The data implicate an in vivo change in mitochondrial permeability in response to victorin. This study focused on whether oat mitochondria can undergo a MPT. Isolated oat mitochondria demonstrated high-amplitude swelling when treated with spermine or Ca2+ in the presence of the Ca2+-ionophore A23187, and when treated with mastoparan, an inducer of the MPT in rat liver mitochondria. In all cases, swelling demonstrated size exclusion in the range 0.9-1.7 kDa, similar to that found in animal mitochondria. Further, MPT-inducing conditions permitted victorin access to the mitochondrial matrix and binding to the P-protein. In vivo, victorin treatment induced the collapse of mitochondrial transmembrane potential within 2 h, indicating a MPT. Also, the victorin-induced collapse of membrane potential was clearly distinct from that induced by uncoupling respiration, as the latter event prevented the victorin-induced PCD response and binding to P-protein. These results demonstrate that a MPT can occur in oat mitochondria in vitro, and are consistent with the hypothesis that an MPT, which allows victorin access to the mitochondrial matrix and binding to the P-protein, occurs in vivo during victorin-induced PCD.     

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.     

Cyclosporin A is unable to inhibit carboxyatractyloside-induced permeability transition in aged mitochondria

We studied the effect of mitochondrial ageing on membrane permeability transition. The results obtained indicate that aged mitochondria are neither able to retain Ca2+ nor to maintain a high transmembrane electric gradient. In addition, aged mitochondria undergo a large amplitude swelling. These dysfunctions were circumvented by the addition of cyclosporin A. Furthermore, it is shown that ageing-induced permeability transition causes oxidative damage on the matrix enzyme aconitase. The observed damage in aged mitochondria requires Ca2+ addition; therefore, it was not seen when Sr2+ replaced Ca2+. Two important findings in this work were the fact that despite of the presence of cyclosporin A, carboxyatractyloside was still able to induce permeability transition, and that ageing induced mitochondrial DNA disruption and release of cytochrome c. It is likely that the membrane's increased permeability is due to the effect of fatty acids, since bovine serum albumin makes mitochondria able to retain Ca2+. However, the possibility that the damage might be the result of oxidative stress cannot be discarded.     

Apoptosis driven by IP(3)-linked mitochondrial calcium signals

Increases of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) evoked by calcium mobilizing agonists play a fundamental role in the physiological control of cellular energy metabolism. Here, we report that apoptotic stimuli induce a switch in mitochondrial calcium signalling at the beginning of the apoptotic process by facilitating Ca(2+)-induced opening of the mitochondrial permeability transition pore (PTP). Thus [Ca(2+)](m) signals evoked by addition of large Ca(2+) pulses or, unexpectedly, by IP(3)-mediated cytosolic [Ca(2+)] spikes trigger mitochondrial permeability transition and, in turn, cytochrome c release. IP(3)-induced opening of PTP is dependent on a privileged Ca(2+) signal transmission from IP(3) receptors to mitochondria. After the decay of Ca(2+) spikes, resealing of PTP occurs allowing mitochondrial metabolism to recover, whereas activation of caspases is triggered by cytochrome c released to the cytosol. This organization provides an efficient mechanism to establish caspase activation while mitochondrial metabolism is maintained to meet ATP requirements of apoptotic cell death.     

Zn(2+) induces permeability transition pore opening and release of pro-apoptotic peptides from neuronal mitochondria

Rapid entry of Ca(2+) or Zn(2+) kills neurons. Mitochondria are major sites of Ca(2+)-dependent toxicity. This study examines Zn(2+)-initiated mitochondrial cell death signaling. 10 nm Zn(2+) induced acute swelling of isolated mitochondria, which was much greater than that induced by higher Ca(2+) levels. Zn(2+) entry into mitochondria was dependent upon the Ca(2+) uniporter, and the consequent swelling resulted from opening of the mitochondrial permeability transition pore. Confocal imaging of intact neurons revealed entry of Zn(2+) (with Ca(2+)) to cause pronounced mitochondrial swelling, which was far greater than that induced by Ca(2+) entry alone. Further experiments compared the abilities of Zn(2+) and Ca(2+) to induce mitochondrial release of cytochrome c (Cyt-c) or apoptosis-inducing factor. In isolated mitochondria, 10 nm Zn(2+) exposures induced Cyt-c release. Induction of Zn(2+) entry into cortical neurons resulted in distinct increases in cytosolic Cyt-c immunolabeling and in cytosolic and nuclear apoptosis-inducing factor labeling within 60 min. In comparison, higher absolute [Ca(2+)](i) rises were less effective in inducing release of these factors. Addition of the mitochondrial permeability transition pore inhibitors cyclosporin A and bongkrekic acid decreased Zn(2+)-dependent release of the factors and attenuated neuronal cell death as assessed by trypan blue staining 5-6 h after the exposures.