Long-chain fatty acids promote opening of the reconstituted mitochondrial permeability transition pore

Adenine nucleotide translocase-porin-hexokinase complex isolated from rat brain, when reconstituted into phospholipid-cholesterol vesicles, exhibits all properties of the mitochondrial permeability transition pore [Beutner, G., Rück, A., Riede, B., Welte, W. and Brdiczka, D. (1996) FEBS Lett. 396, 189-195]. In the present work, the effect of long-chain fatty acids on such reconstituted pore was examined. Opening of the pore was measured by leakage of either malate or fluorescein sulphonate entrapped inside the vesicles. It was found that myristate and oleate in the presence of 50 or 100 microM Ca(2+) produced a partial release of the probes in a dose-dependent way. A dicarboxylic fatty acid analogue, that appeared inactive as protonophore in intact mitochondria, exerted no effect on pore opening in the reconstituted system. 100 microM Ca(2+) alone was without effect. Pore opening by fatty acids in the reconstituted system was partly prevented by cyclosporin A. The pore opening also occurred when the vesicles were incubated in the presence of pancreatic phospholipase A(2). In this case, the opening was decreased by cyclosporin A or serum albumin. These results indicate that long-chain fatty acids elicit opening of the permeability transition pore reconstituted in phospholipid vesicles in a similar way as in intact mitochondria [Wi&ecedil;ckowski, M.R. and Wojtczak, L. (1998) FEBS Lett. 423, 339-342].     

Tetanus, botulinum and snake presynaptic neurotoxins

Tetanus and botulinum neurotoxins, produced by anaerobic bacteria of the genus Clostridium, are the most toxic proteins known and are solely responsible for the pathogenesis of tetanus and botulism. They are metallo-proteases that enter nerve terminals and cleave proteins of the neuroexocytosis apparatus causing a persistent, but reversible, inhibition of neurotransmitter release. Botulinum neurotoxins are used in the therapy of many human syndromes caused by hyperactive nerve terminals. Snake presynaptic PLA2 neurotoxins block nerve terminals by binding to the nerve membrane and catalyzing phospholipid hydrolysis with production of lysophospholipids and fatty acids. These compounds change the membrane conformation causing enhanced fusion of synaptic vesicle via hemifusion intermediate with release of neurotransmitter and, at the same time, inhibition of vesicle fission and recycling. It is possible to envisage clinical applications of the lysophospholipid/fatty acid mixture to inhibit hyperactive superficial nerve terminals.

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How do presynaptic PLA2 neurotoxins block nerve terminals?

Snake presynaptic neurotoxins with phospholipase A2 activity block nerve terminals in an unknown way. Here, we propose that they enter the lumen of synaptic vesicles following endocytosis and hydrolyse phospholipids of the inner leaflet of the membrane. The transmembrane pH gradient drives the translocation of fatty acids to the cytosolic monolayer, leaving lysophospholipids on the lumenal layer. Such vesicles are highly fusogenic and release neurotransmitter upon fusion with the presynaptic membrane, but cannot be retrieved because of the high local concentration of fatty acids and lysophospholipids, which prevents vesicle neck closure.     

Palmitic acid opens a novel cyclosporin A-insensitive pore in the inner mitochondrial membrane

An assortment of agents can induce mitochondria to undergo a permeability transition, which results in the inner mitochondrial membrane becoming nonselectively permeable to small (<1500 Da) solutes. This mitochondrial permeability transition (MPT) is characterized by a strict dependence on matrix Ca2+ and sensitivity to cyclosporin A (CsA). However, it is becoming increasingly clear that other experimental conditions can elicit increases in mitochondrial permeability that are distinct from this classic MPT. For example, butylated hydroxytoluene (BHT; Sokolove, P. M., and Haley, L. M. (1996) J. Bioenerg. Biomembr. 28, 199-206) and signal peptides (Sokolove, P. M., and Kinnally, K. W. (1996) Arch. Biochem. Biophys. 336, 69-76) promote increases in mitochondrial permeability that are CsA-insensitive. It has been suggested (Gudz, T., Eriksson, O., Kushnareva, Y., Saris, N.-E., and Novgorodov, S. A. (1997) Arch. Biochem. Biophys. 342, 143-156) that BHT might be opening a CsA-insensitive pore by increasing phospholipase A2 activity and thereby producing an accumulation of free fatty acids and lysophospholipids. We have therefore examined the effect of the saturated free fatty acid, palmitic acid (PA), on the permeability of isolated rat liver mitochondria. The following results were obtained: (1) In the absence of additional triggers, PA (20-60 microM) induced concentration-dependent, CsA-insensitive mitochondrial swelling. (2) Swelling required mitochondrial energization. (3) PA-induced swelling was fast and occurred without a lag. (4) Both Ca2+ and Sr2+ supported PA-induced swelling; the site of cation action was the matrix. (5) EGTA and BSA were potent inhibitors of PA-induced swelling. (6) PA opened a pore rather than disrupting mitochondrial membrane structure. (7) The pore opened by PA closed spontaneously. These results suggest that palmitic acid promotes a nonclassic permeability increase that is clearly distinguishable from the occurrence of the MPT.     

Dietary supplementation with docosahexaenoic acid, but not eicosapentaenoic acid, dramatically alters cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition

Treatment with the ω-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exerts cardioprotective effects, and suppresses Ca²⁺-induced opening of the mitochondrial permeability transition pore (MPTP). These effects are associated with increased DHA and EPA, and lower arachidonic acid (ARA) in cardiac phospholipids. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, little is known about the independent effects of DHA and EPA on mitochondria function. We compared the effects of dietary supplementation with the ω-3 PUFAs DHA and EPA on cardiac mitochondrial phospholipid fatty acid composition and Ca²⁺-induced MPTP opening. Rats were fed a standard lab diet with either normal low levels of ω-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for Ca²⁺-induced MPTP opening and phospholipid fatty acyl composition. DHA supplementation increased both DHA and EPA and decreased ARA in mitochondrial phospholipid, and significantly delayed MPTP opening as assessed by increased Ca²⁺ retention capacity and decreased Ca²⁺-induced mitochondria swelling. EPA supplementation increased EPA in mitochondrial phospholipids, but did not affect DHA, only modestly lowered ARA, and did not affect MPTP opening. In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition and delayed Ca²⁺-induced MPTP opening.     

Differentiation of phospholipases A in mitochondria and lysosomes of rat liver

Highly purified mitochondria from rat liver contain a phospholipase A that catalyzes removal of 2-fatty acids, with a pH optimum above pH 8.0. Lysosomal preparations appeared to have two phospholipases A associated with them, one with a pH optimum at about pH 4.0, the second between pH 6.0 and 7.0. Mitochondrial phospholipase A hydrolyzed exogenous phospholipid as fast as or faster than endogenous phospholipid. The difference in specific radioactivity of (14)C-ethanolamine-labeled endogenous mitochondrial phospholipid before and after incubation indicates that a fraction of mitochondrial phosphatidyl ethanolamine is hydrolyzed more rapidly than the mitochondrial phospholipids as a whole. Acyl bond hydrolysis of exogenous and endogenous phospholipid by mitochondria was stimulated by free fatty acid, Ca(++), or in certain cases, monoacyl phospholipids or by treatments that disrupt the mitochondrial membrane. Of various fatty acids tested, lauric, myristic, oleic, and linoleic were most effective. ADP and ATP inhibited mitochondrial phospholipase, probably because they compete for Ca(++). Mg(++) also behaved as a competitive inhibitor; the effect was overcome by relatively little Ca(++).     

Swelling of Phaseolus Mitochondria Induced by the Action of Phospholipase A

Phaseolus vulgaris mitochondria incubated in sucrose swell rapidly upon the addition of phospholipase A. Bovine serum albumin inhibits the swelling. The release of free fatty acids as a result of phospholipase A action on the mitochondria is detected only in the presence of bovine serum albumin, which promotes the hydrolysis of both mitochondrial phospholipids and purified lecithin. Either free fatty acid or lysolecithin is able to initiate an extensive mitochondrial swelling in sucrose. It is suggested that phospholipase A-induced swelling results from the release of lysophosphatides plus free fatty acids and their subsequent detergent action on the membranes rather than phospholipid loss per se.     

The effect of Ca2+ and acyl coenzyme A:lysophospholipid acyltransferase inhibitors on permeability properties of the liver mitochondrial inner membrane

We have reported previously that a number of metabolites and toxins which cause Ca2+ release from mitochondria do so by increasing the permeability of the inner membrane. The metabolic basis of this permeability change is proposed to be perturbation of a phospholipid deacylation-reacylation cycle which results in an accumulation of free fatty acids and lysophospholipids (see Broekemeier, K. M., Schmid, P. C., Schmid, H. H. O., and Pfeiffer, D. R. (1985) J. Biol. Chem. 260, 105-113 and references therein). This hypothesis predicts that inhibitors of acyl-CoA:lysophospholipid acyltransferase would be among those agents which increase membrane permeability and that their effects on permeability could occur in the absence of pyridine nucleotide oxidation or of an accumulation of glutathione disulfide. The hypolipidemic drugs WY-14643 and clofibric acid inhibit the mitochondrial acyl-CoA:lysophospholipid acyltransferase and have the predicted effects on mitochondrial permeability properties. The development of increased permeability due to WY-14643 and clofibric acid requires accumulated Ca2+ specifically, is sensitive to inhibitors of phospholipase A2, and results in a pattern of solute release and swelling which is typical of other Ca2+-releasing agents. Neither agent promotes pyridine nucleotide nor sulfhydryl glutathione oxidation in the absence of Ca2+. In addition, the swelling response to hypolipidemic drugs is not significantly inhibited by dithiothreitol. In the presence of Ca2+, both agents promote an accumulation of free fatty acids. The composition of these lipid degradation products suggests that mitochondria treated with hypolipidemic drugs retain an active lysophospholipase whereas this enzyme is inactivated by Ca2+-releasing agents which alter mitochondrial sulfhydryl groups.     

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.     

Phospholipase activity in rat liver mitochondria studied by the use of endogenous substrates

The hydrolysis of endogenous phosphatidyl ethanolamine and lecithin in rat liver mitochondria has been studied by using mitochondria from rats injected with ethanolamine-1,2-(14)C or choline-1,2-(14)C. A phospholipase A-like enzyme has been demonstrated, which catalyzes the hydrolysis of one fatty acid ester linkage in phosphatidyl ethanolamine and lecithin. Phosphatidyl ethanolamine is hydrolyzed in preference to lecithin and the main reaction products are free fatty acids and lysophosphatidyl ethanolamine. The further breakdown of lysophospholipids appears to be limited in mitochondria, which indicates that lysophospholipase activity is mainly located extramitochondrially. The enzymic system is greatly stimulated by calcium ions, and also slightly by magnesium ions, while EDTA inhibits it almost completely. These findings are discussed in relation to previous observations on the effect of calcium and of EDTA on the functions of mitochondria. The possible function of the mitochondrial phospholipase for the formation of phospholipids with special fatty acids at the alpha- and -position is discussed.     

Structural alterations of the inner mitochondrial membrane in ischemic liver cell injury

Mitoplasts were prepared from 3-h ischemic livers in an attempt to define the structural alterations in the inner membrane that may account for the functional deficiencies of ischemic mitochondria. Mitoplasts from both control and ischemic livers had similar specific activities of cytochrome oxidase and succinate-cytochrome c reductase. With both preparations, the specific activity of rotenone-insensitive NADH-cytochrome c reductase was 10-fold lower than in the mitochondria from which they were prepared. Ischemic mitoplasts had no respiratory control with ADP, and had a slightly reduced phospholipid to protein ratio and an increased cholesterol to protein ratio. As a result, the cholesterol to phospholipid molar ratio was increased from the control of 0.04 to 0.08. There were also differences in the content of individual phospholipid species. Phosphatidylcholine increased by 15%, while cardiolipin decreased by 60%. There were increases in sphingomyelin and in the lysophospholipids of phosphatidylcholine, ethanolamine, and cardiolipin. Pretreatment with chlorpromazine did not prevent these changes. Linoleic acid was decreased by 35% in ischemic phospholipids, and the content of free fatty acids was increased 4-fold. Electron spin resonance spectroscopy of mitoplasts spin labeled with either 5- or 12-doxyl stearic acid revealed an increased molecular order (decreased fluidity) of ischemic inner mitochondrial membranes consistent with the increased cholesterol to phospholipid ratio. The data indicate activation of a phospholipase A in ischemic mitochondria with the resulting accumulation of products of lipid hydrolysis. This conclusion further emphasizes the close similarity between the structural and functional consequences of ischemia in the intact animal and the effect on isolated mitochondria of the activation of the endogenous phospholipase A. In both cases the major functional alterations are attributable to changes in the permeability of the inner mitochondrial membrane induced by the accumulation of lysophospholipids.     

Improved mitochondrial function with diet-induced increase in either docosahexaenoic acid or arachidonic acid in membrane phospholipids

Mitochondria can depolarize and trigger cell death through the opening of the mitochondrial permeability transition pore (MPTP). We recently showed that an increase in the long chain n3 polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA; 22:6n3) and depletion of the n6 PUFA arachidonic acid (ARA; 20:4n6) in mitochondrial membranes is associated with a greater Ca(2+) load required to induce MPTP opening. Here we manipulated mitochondrial phospholipid composition by supplementing the diet with DHA, ARA or combined DHA+ARA in rats for 10 weeks. There were no effects on cardiac function, or respiration of isolated mitochondria. Analysis of mitochondrial phospholipids showed DHA supplementation increased DHA and displaced ARA in mitochondrial membranes, while supplementation with ARA or DHA+ARA increased ARA and depleted linoleic acid (18:2n6). Phospholipid analysis revealed a similar pattern, particularly in cardiolipin. Tetralinoleoyl cardiolipin was depleted by 80% with ARA or DHA+ARA supplementation, with linoleic acid side chains replaced by ARA. Both the DHA and ARA groups had delayed Ca(2+)-induced MPTP opening, but the DHA+ARA group was similar to the control diet. In conclusion, alterations in mitochondria membrane phospholipid fatty acid composition caused by dietary DHA or ARA was associated with a greater cumulative Ca(2+) load required to induced MPTP opening. Further, high levels of tetralinoleoyl cardiolipin were not essential for normal mitochondrial function if replaced with very-long chain n3 or n6 PUFAs.     

Evidence suggesting the importance of fatty acids and the fatty acid moieties of sperm membrane phospholipids in the acrosome reaction of guinea pig spermatozoa

When guinea pig spermatozoa were preincubated 1 hr in Ca2+-free medium containing dilysocardiolipin (100--125 micrograms/ml) then exposed to Ca2+, the majority underwent an immediate acrosome reaction. Monolysocardiolipin was much less effective and the native cardiolipin was totally ineffective. Some fatty acids added to the medium could also render the spermatozoa capable of undergoing their acrosome reactions, arachidonic acid in methyl ester form being most potent. It is known that sperm membrane contains phospholipase A which cleaves membrane phospholipids into lysophospholipids and fatty acids. Most lysophospholipids are known to be potent acrosome reaction-promoting agents. As some forms of fatty acids are also potent acrosome reaction-promoting agents, both products of membrane phospholipid hydrolysis by phospholipase A (i.e., both fatty acids and lysophospholipids) may work synergistically to effect the conversion of stable sperm membranes to a fusion competent state capable of engaging in the acrosome reaction.     

Presynaptic enzymatic neurotoxins

Botulinum neurotoxins produced by anaerobic bacteria of the genus Clostridium are the most toxic proteins known, with mouse LD50 values in the 1-5 ng/kg range, and are solely responsible for the pathophysiology of botulism. These metalloproteinases enter peripheral cholinergic nerve terminals and cleave proteins of the neuroexocytosis apparatus, causing a persistent, but reversible, inhibition of neurotransmitter release. They are used in the therapy of many human syndromes caused by hyperactive nerve terminals. Snake presynaptic PLA2 neurotoxins block nerve terminals by binding to the nerve membrane and catalyzing phospholipid hydrolysis with production of lysophospholipids and fatty acids. These compounds change the membrane conformation, causing enhanced fusion of synaptic vesicle via hemifusion intermediate with release of neurotransmitter and, at the same time, inhibition of vesicle fission and recycling. It is possible to envisage clinical applications of the lysophospholipid/fatty acid mixture to inhibit hyperactive superficial nerve terminals.     

On the Role of the Respiratory Complex I on Membrane Permeability Transition

In this work we studied permeability transition by incubating mitochondria in the presence of 50 M Ca²⁺ and malate/glutamate as substrates. This condition, besides inducing the release of pyridine nucleotides, promotes the generation of reactive oxygen-derived species by the complex I of the respiratory chain. The latter leads to the opening of the mitochondrial permeability transition pore. Ca²⁺ release, mitochondrial swelling and collapse of the transmembrane electric potential, were analyzed to assess this process. We propose that the mechanism for pore opening, in addition to the oxidative stress, involves the uncoupling effect of fatty acids providing activation of phospholipase A2, lipid peroxidation, and the oxidation of membrane thiols. This proposal emerges from the data indicating the protective effect of bovine serum albumin and N-ethylmaleimide. The key role of reactive oxygen species was implied based on the fact that the scavenger -phenyl-tert-butyl nitrone inhibited pore opening.     

Effects of fatty acids in isolated mitochondria: implications for ischemic injury and cardioprotection

Fatty acids accumulate during myocardial ischemia and are implicated in ischemia-reperfusion injury and mitochondrial dysfunction. Because functional recovery after ischemia-reperfusion ultimately depends on the ability of the mitochondria to recover membrane potential (DeltaPsim), we studied the effects of fatty acids on DeltaPsim regulation, cytochrome c release, and Ca2+ handling in isolated mitochondria under conditions that mimicked aspects of ischemia-reperfusion. Long-chain but not short-chain free fatty acids caused a progressive and reversible (with BSA) increase in inner membrane leakiness (proton leak), which limited mitochondrial ability to support DeltaPsim. In comparison, long-chain activated fatty acids promoted 1). a slower depolarization that was not reversible with BSA, 2). cytochrome c loss that was unrelated to permeability transition pore opening, and 3). inhibition of the adenine nucleotide translocator. Together, these results impaired both mitochondrial ATP production and Ca2+ handling. Diazoxide, a selective opener of mitochondrial ATP-dependent potassium (KATP) channels, partially protected against these effects. These findings indicate that long-chain fatty acid accumulation during ischemia-reperfusion may predispose mitochondria to cytochrome c loss and irreversible injury and identify a novel cardioprotective action of diazoxide.     

Mitochondrial regulation of synaptic plasticity in the hippocampus

Synaptic mechanisms of plasticity are calcium-dependent processes that are affected by dysfunction of mitochondrial calcium buffering. Recently, we observed that mice deficient in mitochondrial voltage-dependent anion channels, the outer component of the mitochondrial permeability transition pore, have impairments in learning and hippocampal synaptic plasticity, suggesting that the mitochondrial permeability transition pore is involved in hippocampal synaptic plasticity. In this study, we examined the effect on synaptic transmission and plasticity of blocking the permeability transition pore with low doses of cyclosporin A and found a deficit in synaptic plasticity and an increase in base-line synaptic transmission. Calcium imaging of presynaptic terminals revealed a transient increase in the resting calcium concentration immediately upon incubation with cyclosporin A that correlated with the changes in synaptic transmission and plasticity. The effect of cyclosporin A on presynaptic calcium was abolished when mitochondria were depolarized prior to cyclosporin A exposure, and the effects of cyclosporin A and mitochondrial depolarization on presynaptic resting calcium were similar, suggesting a mitochondrial locus of action of cyclosporin A. To further characterize the calcium dynamics of the mitochondrial permeability transition pore, we used an in vitro assay of calcium handling by isolated brain mitochondria. Cyclosporin A-exposed mitochondria buffered calcium more rapidly and subsequently triggered a more rapid mitochondrial depolarization. Similarly, mitochondria lacking the voltage-dependent anion channel 1 isoform depolarized more readily than littermate controls. The data suggest a role for the mitochondrial permeability transition pore and voltage-dependent anion channels in mitochondrial synaptic calcium buffering and in hippocampal synaptic plasticity.     

Sustained oscillations of transmembrane Ca2+ fluxes in mitochondria and their possible biological significance

Sustained oscillations of transmembrane fluxes of Ca2+ and other ions in isolated mitochondria are described. The data are presented that the major cause of the oscillations is the Ca2+-induced Ca2+ efflux from the mitochondrial matrix and spontaneous opening/closing of the permeability transition pore in the inner mitochondrial membrane. Conditions favourable for the generation of oscillations are considered. The role of phospholipid peroxidation and hydrolysis in the generation of [Ca2+] oscillations is emphasized. Literature data concerning [Ca2+] changes in the mitochondrial matrix in intact cells and the data on the participation of mitochondria in intracellular Ca2+ oscillation and in the Ca2+ wave propagation are reviewed. The hypothesis that mitochondria are able to generate [Ca2+] oscillations in intact cells is put forward. It is assumed that Ca2+ oscillations can protect mitochondria of resting cells from osmotic shock and oxidative stress.     

Cytochrome c release precedes mitochondrial membrane potential loss in cerebellar granule neuron apoptosis: lack of mitochondrial swelling

It has been suggested that release of cytochrome c (Cyt c) from mitochondria during apoptotic death is through opening of the mitochondrial permeability transition pore followed by swelling-induced rupture of the mitochondrial outer membrane. However, this remains controversial and may vary with cell type and model system. We determined that in mouse cerebellar granule neurons, Cyt c redistribution preceded the loss of mitochondrial membrane potential during the apoptotic process, suggesting that the pore did not open prior to release. Furthermore, when mitochondria were morphologically assessed by electron microscopy, they were not obviously swollen during the period of Cyt c release. This indicates that the pore mechanism of action, if any, is not through mitochondrial outer membrane rupture. While bongkrekic acid, an inhibitor of pore opening, modestly delayed apoptotic death, it also caused a significant (p < 0.05) suppression of protein synthesis. An equivalent suppression of protein synthesis by cycloheximide had a similar delaying effect, suggesting that bongkrekic acid was acting non-specifically. These findings suggest that mitochondrial permeability transition pore is not involved in Cyt c release from mitochondria during the apoptotic death of cerebellar granule neurons.     

Role of mitochondrial membrane lipid hydrolysis in their swelling induced by thyroxine]

The thyroxin-induced mitochondrial swelling was accompanied by an accumulation in organellas of free fatty acids which level was restored after the mitochondria contraction in the ATP presence. EGTA induced mitochondrial contractions as well, but with no free fatty acids utilization. Apparently, the thyroxin-induced mitochondrial swelling is the result of the membrane phospholipase activation and of the increase in the membrane cationic permeability due to the hydrolysis of membrane phospholipids.