Ca2+ handling in isolated brain mitochondria and cultured neurons derived from the YAC128 mouse model of Huntington's disease

We investigated Ca²⁺ handling in isolated brain synaptic and non‐synaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington's disease. Both synaptic and non‐synaptic mitochondria from 2‐ and 12‐month‐old YAC128 mice had larger Ca²⁺ uptake capacity than mitochondria from YAC18 and wild‐type FVB/NJ mice. Synaptic mitochondria from 12‐month‐old YAC128 mice had further augmented Ca²⁺ capacity compared with mitochondria from 2‐month‐old YAC128 mice and age‐matched YAC18 and FVB/NJ mice. This increase in Ca²⁺ uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12‐month‐old YAC128 mice. We speculate that this may happen because of mHtt‐mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca²⁺‐induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100 μM glutamate produced transient elevations in cytosolic Ca²⁺ followed by recovery to near resting levels. Following recovery of cytosolic Ca²⁺, mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca²⁺, suggesting similar Ca²⁺ release and, consequently, Ca²⁺ loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca²⁺ handling in brain mitochondria of YAC128 mice.

     

Mitochondrial permeability transition in Ca(2+)-dependent apoptosis and necrosis

A variety of stimuli utilize an increase of cytosolic free Ca²⁺ concentration as a second messenger to transmit signals, through Ca²⁺ release from the endoplasmic reticulum or opening of plasma membrane Ca²⁺ channels. Mitochondria contribute to the tight spatiotemporal control of this process by accumulating Ca²⁺, thus shaping the return of cytosolic Ca²⁺ to resting levels. The rise of mitochondrial matrix free Ca²⁺ concentration stimulates oxidative metabolism; yet, in the presence of a variety of sensitizing factors of pathophysiological relevance, the matrix Ca²⁺ increase can also lead to opening of the permeability transition pore (PTP), a high conductance inner membrane channel. While transient openings may serve the purpose of providing a fast Ca²⁺ release mechanism, persistent PTP opening is followed by deregulated release of matrix Ca²⁺, termination of oxidative phosphorylation, matrix swelling with inner membrane unfolding and eventually outer membrane rupture with release of apoptogenic proteins and cell death. Thus, a rise in mitochondrial Ca²⁺ can convey both apoptotic and necrotic death signals by inducing opening of the PTP. Understanding the signalling networks that govern changes in mitochondrial free Ca²⁺ concentration, their interplay with Ca²⁺ signalling in other subcellular compartments, and regulation of PTP has important implications in the fine comprehension of the main biological routines of the cell and in disease pathogenesis.     

α-Synuclein induced membrane depolarization and loss of phosphorylation capacity of isolated rat brain mitochondria: Implications in Parkinson’s disease

This study demonstrates that in vitro incubation of isolated rat brain mitochondria with recombinant human α-synuclein leads to dose-dependent loss of mitochondrial transmembrane potential and phosphorylation capacity. However, α-synuclein does not seem to have any significant effect on the activities of respiratory chain complexes under similar conditions of incubation suggesting that the former may impair mitochondrial bioenergetics by direct effect on mitochondrial membranes. Moreover, the recombinant wild type α-synuclein and different mutant forms (A30P, A53T and E46K) have essentially similar effects on rat brain isolated mitochondria. The results are significant in view of the fact that α-synucleinopathy is involved in the pathogenesis of Parkinson’s disease.     

Ascorbate and low concentrations of FeSO4 induce Ca2+-dependent pore in rat liver mitochondria

Oxidative stress is one of the most frequent causes of tissue and cell injury in various pathologies. The molecular mechanism of mitochondrial damage under conditions of oxidative stress induced in vitro with low concentrations of FeSO₄ and ascorbate (vitamin C) was studied. FeSO₄ (1-4 M) added to rat liver mitochondria that were incubated in the presence of 2.3 mM ascorbate induced (with a certain delay) a decrease in membrane potential and high-amplitude swelling. It also significantly decreased the ability of mitochondria to accumulate exogenous Ca²⁺. All the effects of FeSO₄ + ascorbate were essentially prevented by cyclosporin A, a specific inhibitor of the mitochondrial Ca²⁺-dependent pore (also known as the mitochondrial permeability transition). EGTA restored the membrane potential of mitochondria de-energized with FeSO₄ + ascorbate. We hypothesize that oxidative stress induced in vitro with FeSO₄ and millimolar concentrations of ascorbate damages mitochondria by inducing the cyclosporin A-sensitive Ca²⁺-dependent pore in the inner mitochondrial membrane.     

Complex contribution of cyclophilin D to Ca2+-induced permeability transition in brain mitochondria, with relation to the bioenergetic state

Cyclophilin D (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca(2+), and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca(2+) caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca(2+) in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca(2+) deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli.     

Intracellular Ca2+ signaling and human disease: the hunt begins with Huntington's

Huntingtin, a protein altered by polyglutamine expansion in Huntington's disease (Httexp), forms a signaling complex with the InsP3R, an intracellular calcium channel, and Htt-associated protein 1A (HAP1A). The addition of Httexp increases the InsP3R sensitivity to InsP3, which subsequently makes neurons hyperresponsive to stimulation and presumably more prone to neurodegenerative processes.     

Disruption of the toxic conformation of the expanded polyglutamine stretch leads to suppression of aggregate formation and cytotoxicity

The polyglutamine (polyQ) diseases are a class of inherited neurodegenerative diseases including Huntington's disease, caused by the expansion of a polyQ stretch within each disease protein. This expansion is thought to cause a conformational change in the protein leading to aggregation of the protein, resulting in cytotoxicity. To analyze whether disrupting the toxic conformation of the polyQ protein can alter its aggregation propensity and cytotoxicity, we examined the effect of interruption of the expanded polyQ stretch by proline insertion, since prolines cause great alterations in protein conformation. Here, we show that insertion of prolines into the expanded polyQ stretch indeed disrupts its ordered secondary structure, leading to suppression of polyQ protein aggregation both in vitro and in cell culture, and reduction of cytotoxicity in correlation with the number of proline interruptions. Furthermore, we found that a short polyQ stretch with a proline interruption is able to inhibit aggregation of the expanded polyQ protein in trans. These results show that a gain in toxic conformation of the expanded polyQ protein is essential for aggregation and cytotoxicity, providing insight into establishing therapies against the polyQ diseases.     

The Ca2+-dependent protease inhibitor of rat ventral prostate: properties of the inhibitor and effects of castration on Ca2+-dependent protease and inhibitor activities

1. The rat ventral prostate contains a heat stable inhibitor of Ca2+-dependent protease. This inhibitor was found to exist in a wide range of molecular weights (approx. 40-270 kDa) in adult rats. 2. However, in rats immediately post puberty (45 days of age) the inhibitor was predominantly of the higher molecular weight forms. 3. The inhibitor was also found in the dorsolateral and anterior (coagulating gland) prostate lobes but was of lower specific activity than in the ventral lobe. 4. Although the activities of the Ca2+-dependent protease and inhibitor decreased per ventral prostate gland after castration, these activities were not different during the first 10 days postcastration when expressed per g wet wt or per unit cytosol protein. 5. With a longer duration of castration, there was a decline in the specific activity (per unit protein) of the protease and an increase in that of the inhibitor. 6. Thus, the activities of the protease and inhibitor change in concert with the amount of cellular cytosol protein during the active period of castration-induced atrophy. 7. However, in long term castrated rats, functions carried out by the Ca2+-dependent protease may be effectively suppressed. 8. These data suggest that the Ca2+-activated protease probably is involved in the regulation of some metabolic processes in the active gland and is not prominent in the castration induced atrophy of the ventral prostate unless it functions through the proteolysis of some select protein(s).     

In vitro effects of microwave radiation on rat liver mitochondria

Liver mitochondria were exposed in vitro at 30°C to microwave radiation (2.45 GHz) during the following states of respiraton: resting, state 1; substrate dependent, state 2; ADP stimulated, state 3; and ADP depleted, state 4. At 10 or 100 mW/g, with succinate as substrate, no effect of exposure was observed on states 1–4 or the respiratory control index (state 3/state 4) of either tightly or loosely coupled mitochondria. When glutamate was used as substrate, no effects were observed at 10 mW/g. However, in the loosely coupled mitochondria the 100 mW/g exposure produced an increase in states 2 and 4 and a decrease in the respiratory control index. The results suggest that the function of loosely coupled mitochondria can be affected at high power levels of microwave radiation.     

Role of mitochondria in the immune response to cancer: a central role for Ca2+

This study demonstrates that Ca²⁺ stimulates mitochondrial energy metabolism during spleen lymphocyte activation in response to the ascitic Walker 256 tumor in rats. Intracellular Ca²⁺ concentrations, phosphorylated protein kinase C (pPKC) levels, Bcl-2 protein contents, interleukin-2 (IL-2) levels, mitochondrial uncoupling protein-2 (UCP-2) contents and reactive oxygen species (ROS) were significantly elevated in these activated lymphocytes. Mitochondria of activated lymphocytes exhibited high free Ca²⁺ concentrations in the matrix and enhanced oligomycin-sensitive oxygen consumption, indicating an increased rate of oxidative phosphorylation. The production of ROS was largely decreased by diphenylene iodinium in the activated lymphocytes, suggesting that NADPH oxidase is the prevalent source of these species. Accumulation of UCP-2 and the anti-apoptotic protein Bcl-2 is probably important to prevent mitochondrial dysfunction and cell death elicited by the sustained high levels of intracellular Ca²⁺ and ROS and may explain the observed higher resistance from activated lymphocytes against the opening of the mitochondrial membrane permeability pore (MPT). All these changes were blocked by pretreatment of the rats with verapamil, an L-type Ca²⁺ channel antagonist. These data demonstrate a central role of Ca²⁺ in the control of mitochondrial bioenergetics in spleen lymphocytes during the immune response to cancer.     

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria.

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.     

Regulation of Ca2+ fluxes in rat liver mitochondria by Ca2+. Effects on Ca2+ distribution

Rat liver mitochondria are able to temporarily lower the steady-state concentration of external Ca²⁺ after having accumulated a pulse of added Ca²⁺. This has been attributed to inhibition of a putative -modulated efflux pathway [Bernardi, P. (1984)Biochim. Biophys. Acta 766, 277–282]. On the other hand, the rebounding could be due to stimulation of the uniporter by Ca²⁺ [Kröner, H. (1987)Biol. Chem. Hoppe-Seyler 369, 149–155]. By measuring unidirectional Ca²⁺ fluxes, it was found that the uniporter was stimulated during the rebounding peak both under Bernardi's and Kröner's conditions, while no effects on the efflux could be demonstrated. The rate of unidirectional efflux of Ca²⁺ was not affected by inhibition of the uniporter. It appears likely that the rebounding is due to stimulation of the uniporter rather than inhibition of efflux.     

Studies of Ca2+ -dependent smooth muscle mitochondria swelling using flow cytometry and spermine effects on this process

In the experiments, which were carried out using flow cytometry on isolated uterus mitochondria of nonpregnant rats, conditions for studying Ca2+-induced increase in nonspecific permeability of mitochondial membrane were tested. Fluorescent probe nonyl acridin orange was used to determine the purity of isolated mitochondria. Mitochondrial swelling induced by addition of Ca2+ or alamethicin was detected as a decrease in light side scattering (SS). It was shown that mitochondrial swelling in the presence of 100 microM Ca plus 1 microM A23187 was 86 +/- 4% compared with maximal response after alamethicin treatment. The calcium-induced swelling of mitochondria was inhibited by the addition of 5 microM cyclosporin plus 40 microM ADP. Mitochondrial swelling was inhibited by spermine at a dose of 0.1 micromol/mg or induced at a dose of 10 micromol/mg. It was supposed that the experimental approach proposed in this paper can be useful for mitochondrial pore modulating effectors screening.     

Cd2+ versus Ca2+-produced mitochondrial membrane permeabilization: a proposed direct participation of respiratory complexes I and III

A comparison of Cd²⁺ and Ca²⁺ effects on in vitro rat liver mitochondria function and a further study of their interaction were conducted. Similarity and distinction in action of rotenone, oligomycin, N-ethylmaleimide, dithiothreitol, catalase, dibucaine, ruthenium red, cyclosporin A (CsA), and ADP on Cd²⁺ and/or Ca²⁺-induced mitochondrial dysfunction were revealed. We found that rotenone exerted a strong protective action both against Ca²⁺ and Cd²⁺-produced mitochondrial membrane permeabilization (MMP). In contrast to Ca²⁺, catalase and dibucaine did not influence on main Cd²⁺ effects. In NH₄NO₃ medium N-ethylmaleimide (NEM) at low concentrations increased markedly Cd²⁺-produced swelling of non-energized mitochondria, whereas it exhibited a partial reversal effect following energization. In sucrose medium low [NEM] did not change Cd²⁺-produced mitochondrial swelling. High [NEM] promoted synergistic increase of the Cd²⁺-produced swelling in NH₄NO₃ medium; all above effects were reversed (and prevented) by dithiothreitol, DTT. We shown also that when exogenous Ca²⁺ and P_i were simultaneously present in NH₄NO₃ medium, DTT reversed only partially Cd²⁺-produced swelling of succinate plus rotenone-energized mitochondria, while DTT recovery action was complete when either Ca²⁺ or P_i were separately administered to the Cd²⁺-treated mitochondria. Besides, DTT added following a low Cd²⁺ pulse in KCl medium containing exogenous Ca²⁺ induced a substantial enhancing of sustained Cd²⁺ stimulation of mitochondrial basal respiration and the stimulation was CsA-sensitive, while the activation promoted by low [Cd²⁺] alone was totally eliminated by DTT supplement. We observed the similar respiratory activation earlier when high concentrations of Cd²⁺ in the absence of added Ca²⁺ were used but it was completely CsA-insensitive. A possible involvement of respiratory chain components, namely complex I (P-site) and complex III (S-site) in Cd²⁺and/or Ca²⁺-produced MMP was discussed.     

Effect of pH and Ca2+ on the retention of Ca2+ by rat liver mitochondria.

A transient Ca²⁺ release from preloaded mitochondria can be induced by a sudden decrease in the pH of the outer medium from 8.0 or 7.4 to 6.8. In the presence of inorganic phosphate the released Ca²⁺ is not taken up again. Upon Ca²⁺ addition to respiring mitochondria the mitochondrial membrane potential (Δ♀) decreases to a new resting level. A further decrease in Δ♀ occurs after the decrease in pH from 7.4 to 6.8, concomitant with the reuptake phase of the Ca²⁺ release. Phosphate, EGTA, and ruthenium red restore Δ♀ to its initial level. If phosphate is present initially, only transient changes in Δ♀ occur upon addition of Ca²⁺ or H⁺ ions. Only a small transient change in Δ♀ upon H⁺ ion addition is seen in the absence of accumulated Ca²⁺. La³⁺, a competitive inhibitor of Ca²⁺ transport, prevents the H⁺ ion-induced Ca²⁺ efflux, whereas this is not the case in the presence of the noncompetitive inhibitor ruthenium red. Ruthenium red, however, prevents the reuptake phase. Mg²⁺, an inhibitor of the surface binding of Ca²⁺, has no or only a slight effect on the H⁺ ion-induced Ca²⁺ release. Mitochondria preloaded with Ca²⁺ release a small fraction of Ca²⁺ during the subsequent uptake of another pulse of Ca²⁺. The results indicate that at least one pool of mitochondrial Ca²⁺ exists in a mobile state. The possible existence of a $\text{H}{}^{\mathrm{+}}\text{Ca}{}^{\mathrm{2+}}$ exchanger in the mitochondrial membrane is discussed.     

Role of methionine 35 in the intracellular Ca2+ homeostasis dysregulation and Ca2+-dependent apoptosis induced by amyloid beta-peptide in human neuroblastoma IMR32 cells

Amyloid beta-peptide (Abeta) plays a fundamental role in the pathogenesis of Alzheimer's disease. We recently reported that the redox state of the methionine residue in position 35 of amyloid beta-peptide (Abeta) 1-42 (Met35) strongly affects the peptide's ability to trigger apoptosis and is thus a major determinant of its neurotoxicity. Dysregulation of intracellular Ca(2+) homeostasis resulting in the activation of pro-apoptotic pathways has been proposed as a mechanism underlying Abeta toxicity. Therefore, we investigated correlations between the Met35 redox state, Abeta toxicity, and altered intracellular Ca(2+) signaling in human neuroblastoma IMR32 cells. Cells incubated for 6-24 h with 10 microM Abeta1-42 exhibited significantly increased KCl-induced Ca(2+) transient amplitudes and resting free Ca(2+) concentrations. Nifedipine-sensitive Ca(2+) current densities and Ca(v)1 channel expression were markedly enhanced by Abeta1-42. None of these effects were observed when cells were exposed to Abeta containing oxidized Met35 (Abeta1-42(Met35-Ox)). Cell pre-treatment with the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (1 microM) or the Ca(v)1 channel blocker nifedipine (5 microM) significantly attenuated Abeta1-42-induced apoptosis but had no effect on Abeta1-42(Met35-Ox) toxicity. Collectively, these data suggest that reduced Met35 plays a critical role in Abeta1-42 toxicity by rendering the peptide capable of disrupting intracellular Ca(2+) homeostasis and thereby provoking apoptotic cell death.     

Biodegradable delivery system containing a peptide inhibitor of polyglutamine aggregation: a step toward therapeutic development in Huntington's disease

Huntington's and eight other neurodegenerative diseases occur because of CAG repeat expansion mutation culminating into an expanded polyglutamine tract in respective protein. In Huntington's disease (HD), a number of CAG repeats beyond normal repeat length (>36) lead to the formation of mutant protein, the proteolytic cleavage of which induces aggregation in polyglutamine length‐dependent manner. The neurodegeneration in this disease is linked to aggregation, and its inhibition is a potential approach for therapeutic development. Although peptides and other molecules have been developed for inhibiting aggregation, peptides in general are susceptible to degradation in vivo conditions. To understand their clinical significance, they also need to be delivered through blood–brain barrier. Here, for the first time, we have synthesized poly‐d ,l ‐lactide‐co‐glycolide nanoparticles containing a polyglutamine aggregation inhibitor peptide PGQ₉[P²], by nanoprecipitation method. This process yielded less than 200 nm spherical nanoparticles with uniform distribution. Characterization studies by infrared spectroscopy‐based and HPLC‐based assays show the presence of PGQ₉[P²] in nanoparticles. In vitro release kinetics demonstrates that nanoparticles release PGQ₉[P²] by erosion and diffusion processes. When the PGQ₉[P²]‐loaded nanoparticles are incubated with aggregation‐prone Q₃₅P₁₀ peptide, representing N‐terminal part of Huntingtin protein, it arrests the elongation phase of Q₃₅P₁₀ aggregation. These findings propose the first step toward delivery of a peptide inhibitor against polyglutamine aggregation in HD. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Two mutations in mitochondrial ATP6 gene of ATP synthase,related to human cancer,affect ROS,calcium homeostasis and mitochondrial permeability transition in yeast

The relevance of mitochondrial DNA (mtDNA) mutations in cancer process is still unknown. Since the mutagenesis of mitochondrial genome in mammals is not possible yet, we have exploited budding yeast S. cerevisiae as a model to study the effects of tumor-associated mutations in the mitochondrial MTATP6 gene, encoding subunit 6 of ATP synthase, on the energy metabolism. We previously reported that four mutations in this gene have a limited impact on the production of cellular energy. Here we show that two mutations, Atp6-P163S and Atp6-K90E (human MTATP6-P136S and MTATP6-K64E, found in prostate and thyroid cancer samples, respectively), increase sensitivity of yeast cells both to compounds inducing oxidative stress and to high concentrations of calcium ions in the medium, when Om45p, the component of porin complex in outer mitochondrial membrane (OM), was fused to GFP. In OM45-GFP background, these mutations affect the activation of yeast permeability transition pore (yPTP, also called YMUC, yeast mitochondrial unspecific channel) upon calcium induction. Moreover, we show that calcium addition to isolated mitochondria heavily induced the formation of ATP synthase dimers and oligomers, recently proposed to form the core of PTP, which was slower in the mutants. We show the genetic evidence for involvement of mitochondrial ATP synthase in calcium homeostasis and permeability transition in yeast. This paper is a first to show, although in yeast model organism, that mitochondrial ATP synthase mutations, which accumulate during carcinogenesis process, may be significant for cancer cell escape from apoptosis.     

On the mechanism of palmitic acid-induced apoptosis: the role of a pore induced by palmitic acid and Ca2+ in mitochondria

Palmitic acid (Pal) is known to promote apoptosis (Sparagna G et al (2000) Am J Physiol Heart Circ Physiol 279: H2124–H2132) and its amount in blood and mitochondria increases under some pathological conditions. Yet, the mechanism of the proapoptotic action of Pal has not been elucidated. We present evidence for the involvement of the mitochondrial cyclosporin A-insensitive pore induced by Pal/Ca²⁺ complexes in the apoptotic process. Opening of this pore led to a fall of the mitochondrial membrane potential and the release of the proapoptotic signal cytochrome c. The addition of cytochrome c prevented these effects and recovered membrane potential, which is in contrast to the cyclosporin A-sensitive mitochondrial permeability transition pore. Oleic and linoleic acids prevented the Pal/Ca²⁺-induced pore opening in the intact mitochondria, this directly and significantly correlating with the effect of these fatty acids on Pal-induced apoptosis in cells (Hardy S et al (2003) J Biol Chem 278: 31861–31870). The specific probe for cardiolipin, 10-N-nonyl acridine orange, inhibited formation of this pore.