The iPLA(2)γ is identified as the membrane potential sensitive phospholipase in liver mitochondria.

Previous reports from our lab identified a mitochondrial calcium-independent phospholipase A2 activity that is activated when the mitochondrial membrane potential is decreased. This activity was demonstrated to influence occurrence of the permeability transition. Originally, this activity was ascribed to the iPLA2ß protein. Recently, both iPLA2ß and iPLA2γ knock out mice have been generated. It has been shown by others that the iPLA2γ plays a significant role in progression of the permeability transition. In this paper, using the iPLA2ß and iPLA2γ knock out mice we show that the membrane potential sensitive activity is the iPLA2γ.

The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase.

Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein (LpYhhN), and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a Vmax of 12 µmol/min/mg protein and a Km of 45 µM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5-7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host.

Does oral supplementation of a fermented papaya preparation correct respiratory burst function of innate immune cells in type 2 diabetes mellitus patients?

Fermented papaya preparation (FPP) is a nutritional supplement reported to act as an antioxidant by scavenging reactive oxygen species (ROS) and removing "bad ROS," while inducing "respiratory burst" production of necessary "good ROS." We sought to investigate the safety of oral administration of FPP (9 g/day, 6 weeks) to T2D patients with regard to its effect on the hyperglycemia status of these patients. Peripheral blood was collected during a baseline visit, followed by subsequent collections both during and after supplementation. Induced "respiratory burst" ROS production was measured at each visit in addition to fasting blood glucose, lipid profile, glycated hemoglobin (HbA1c), and lipid/protein peroxidation. Oral FPP supplementation induced "respiratory burst" in peripheral blood mononuclear cells while not influencing other blood parameters studied. When human monocytic THP-1 cells were supplemented with sugar-based FPP, cellular ATP and NADPH concentrations were increased while matched glucose alone did not produce similar effects, suggesting a glucose-independent component of FPP to be responsible for increasing cellular energetics. THP-1 cells supplemented with FPP also exhibited higher mitochondrial membrane potential (Δψm) and oxygen consumption as compared with cells treated with glucose alone. Taken together, our observations lead to the hypothesis that FPP corrects inducible "respiratory burst" function in type 2 diabetes patients.

Mitochondrial uncoupling does not decrease reactive oxygen species production after ischemia-reperfusion.

Cardiac ischemia-reperfusion (IR) leads to myocardial dysfunction by increasing production of reactive oxygen species (ROS). Mitochondrial H(+) leak decreases ROS formation; it has been postulated that increasing H(+) leak may be a mechanism of decreasing ROS production after IR. Ischemic preconditioning (IPC) decreases ROS formation after IR, but the mechanism is unknown. We hypothesize that pharmacologically increasing mitochondrial H(+) leak would decrease ROS production after IR. We further hypothesize that IPC would be associated with an increase in the rate of H(+) leak. Isolated male Sprague-Dawley rat hearts were subjected to either control or IPC. Mitochondria were isolated at end equilibration, end ischemia, and end reperfusion. Mitochondrial membrane potential (mΔΨ) was measured using a tetraphenylphosphonium electrode. Mitochondrial uncoupling was achieved by adding increasing concentrations of FCCP. Mitochondrial ROS production was measured by fluorometry using Amplex-Red. Pyridine dinucleotide levels were measured using HPLC. Before IR, increasing H(+) leak decreased mitochondrial ROS production. After IR, ROS production was not affected by increasing H(+) leak. H(+) leak increased at end ischemia in control mitochondria. IPC mitochondria showed no change in the rate of H(+) leak throughout IR. NADPH levels decreased after IR in both IPC and control mitochondria while NADH increased. Pharmacologically, increasing H(+) leak is not a method of decreasing ROS production after IR. Replenishing the NADPH pool may be a means of scavenging the excess ROS thereby attenuating oxidative damage after IR.

Regulation of the Ca(2+)-independent phospholipase A2 in liver mitochondria by changes in the energetic state.

The effect of electron transport chain redox status on activity of the mitochondrial Ca(2+)-independent phospholipase A2 (iPLA2) has been examined. When oxidizing NAD-linked substrates, the enzyme is not active unless deenergization occurs. Uncoupler, rotenone, antimycin A, and cyanide are equally effective at upregulating the enzyme, while oligomycin is ineffective. Thenoyltrifluoroacetone causes deenergization and activates the enzyme, but only if succinate is the respiratory substrate. These findings show that the mitochondrial iPLA2 responds to the energetic state overall, rather than to the redox status of individual electron transport chain complexes. With NAD-linked substrates, and using rotenone to deenergize, iPLA2 activation can be reversed by adding succinate to reestablish a membrane potential. For this purpose, ascorbate plus N,N,N'N'-tetramethyl-phenylenediamine can be used instead of succinate and is equally effective. With succinate as substrate, the membrane potential can be reduced in a graded and stable fashion by adding increasing concentrations of malonate, which is a competitive inhibitor of succinate utilization. A partial and stable activation of the iPLA2 accompanies partial deenergization. These findings suggest that in addition to the several functions that have been proposed, the mitochondrial iPLA2 may help to coordinate local capillary blood flow with changing energy demands.

Characterization of the respiration-induced yeast mitochondrial permeability transition pore.

When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane, dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable through the yPTP in an industrial yeast strain, Yeast Foam. The presence of oligomycin, an inhibitor of ATP synthase, allowed for respiration-induced mannitol permeability in mitochondria from this strain. Potassium (K+) had varied effects on the respiration-induced yPTP, depending on the concentration of the respiratory substrate added. At low respiratory substrate concentrations K+ inhibited respiration-induced yPTP opening, while at high substrate concentrations this effect diminished. However, at the high respiratory substrate concentrations, the presence of K+ partially prevented phosphate inhibition of yPTP opening. Phosphate was found to inhibit respiration-induced yPTP opening by binding a site on the matrix space side of the inner membrane in addition to its known inhibitory effect of donating protons to the matrix space to prevent the pH change necessary for yPTP opening. The respiration-induced yPTP was also inhibited by NAD, Mg2+, NH4 + or the oxyanion vanadate polymerized to decavanadate. The results demonstrate similar effectors of the respiration-induced yPTP as those previously described for the ATP-induced yPTP and reconcile previous strain-dependent differences in yPTP solute selectivity.

Ischemic preconditioning preserves mitochondrial membrane potential and limits reactive oxygen species production.

BACKGROUND: Mitochondrial superoxide radical (O(2)(•¯)) production increases after cardiac ischemia/reperfusion (IR). Ischemic preconditioning (IPC) preserves mitochondrial function and attenuates O(2)(•¯) production, but the mechanism is unknown. Mitochondrial membrane potential (mΔΨ) is known to affect O(2)(•¯) production; mitochondrial depolarization decreases O(2)(•¯) formation. We examined the relationship between O(2)(•¯) production and mΔΨ during IR and IPC. MATERIALS/METHODS: Rat hearts were subjected to Control or IPC. Mitochondria were isolated at end equilibration (End EQ), end ischemia (End I), and end reperfusion (End RP). mΔΨ was measured using a tetraphenylphosphonium electrode. Mitochondrial O(2)(•¯) production was measured by electron paramagnetic resonance using DMPO spin trap. Cytochrome c levels were measured using high-pressure liquid chromatography. RESULTS: IPC preserved mΔΨ at End I (-156 ± 5 versus -131 ± 6 mV, P < 0.001) and End RP (-168 ± 2 versus -155 ± 2 mV, P < 0.05). At End RP, IPC attenuated O(2)(•¯) production (2527 ± 221 versus 3523 ± 250 AU/mg protein, P < 0.05). IPC preserved cytochrome c levels (351 ± 14 versus 269 ± 16 picomoles/mg protein, P < 0.05) at End RP, and decreased mitochondrial cristae disruption (10% ± 4% versus 33% ± 7%, P < 0.05) and amorphous density formation (18% ± 4% versus 28% ± 1%, P < 0.05). CONCLUSION: We conclude that IPC preserves mΔΨ, possibly by limiting disruption of mitochondrial inner membrane. IPC also decreases mitochondrial O(2)(•¯) production and preserves mitochondrial ultrastructure after IR. While it was previously held that slight decreases in mΔΨ decrease O(2)(•¯) production, our results indicate that preservation of mΔΨ is associated with decreased O(2)(•¯) and preservation of cardiac function in IPC. These findings indicate that the mechanism of IPC may not involve mΔΨ depolarization, but rather preservation of mitochondrial electrochemical potential.

Mitochondrial dynamics and motility inside living vascular endothelial cells: role of bioenergetics.

The mitochondrial network is dynamic with conformations that vary between a tubular continuum and a fragmented state. The equilibrium between mitochondrial fusion/fission, as well as the organelle motility, determine network morphology and ultimately mitochondrial/cell function. Network morphology has been linked with the energy state in different cell types. In this study, we examined how bioenergetic factors affect mitochondrial dynamics/motility in cultured vascular endothelial cells (ECs). ECs were transduced with mitochondria-targeted green fluorescent protein (mito-GFP) and exposed to inhibitors of oxidative phosphorylation (OXPHOS) or ATP synthesis. Time-lapse fluorescence videos were acquired and a mathematical program that calculates size and speed of each mitochondrial object at each time frame was developed. Our data showed that inner mitochondrial membrane potential (ΔΨ(m)), ATP produced by glycolysis, and, to a lesser degree, ATP produced by mitochondria are critical for maintaining the mitochondrial network, and different metabolic stresses induce distinct morphological patterns (e.g., mitochondrial depolarization is necessary for "donut" formation). Mitochondrial movement, characterized by Brownian diffusion with occasional bursts in displacement magnitude, was inhibited under the same conditions that resulted in increased fission. Hence, imaging/mathematical analysis shed light on the relationship between bioenergetics and mitochondrial network morphology; the latter may determine EC survival under metabolic stress.

Purification, identification, and cloning of lysoplasmalogenase, the enzyme that catalyzes hydrolysis of the vinyl ether bond of lysoplasmalogen.

Lysoplasmalogenase (EC 3.3.2.2 and EC 3.3.2.5) is an enzyme that catalyzes hydrolytic cleavage of the vinyl ether bond of lysoplasmalogen, forming fatty aldehyde and glycerophosphoethanolamine or glycerophosphocholine and is specific for the sn-2-deacylated form of plasmalogen. Here we report the purification, characterization, identification, and cloning of lysoplasmalogenase. Rat liver microsomal lysoplasmalogenase was solubilized with octyl glucoside and purified 500-fold to near homogeneity using four chromatography steps. The purified enzyme has apparent K(m) values of ∼50 µm for both lysoplasmenylcholine and lysoplasmenylethanolamine and apparent V(m) values of 24.5 and 17.5 µmol/min/mg protein for the two substrates, respectively. The pH optimum was 7.0. Lysoplasmalogenase was competitively inhibited by lysophosphatidic acid (K(i) ∼20 µm). The predominant band on a gel at ∼19 kDa was subjected to trypsinolysis, and the peptides were identified by mass spectrometry as Tmem86b, a protein of unknown function. Transient transfection of human embryonic kidney (HEK) 293T cells showed that TMEM86b cDNA yielded lysoplasmalogenase activity, and Western blot analyses confirmed the synthesis of TMEM86b protein. The protein was localized in the membrane fractions. The TMEM86b gene was also transformed into Escherichia coli, and its expression was verified by Western blot and activity analyses. Tmem86b is a hydrophobic transmembrane protein of the YhhN family. Northern blot analyses demonstrated that liver expressed the highest level of Tmem86b, which agreed with tissue distribution of activity. Overexpression of TMEM86b in HEK 293T cells resulted in decreased levels of plasmalogens, suggesting that the enzyme may be important in regulating plasmalogen levels in animal cells.

Ischemic preconditioning decreases mitochondrial proton leak and reactive oxygen species production in the postischemic heart.

BACKGROUND: Proton leak (H(+) leak) dissipates mitochondrial membrane potential (mΔΨ) through the re-entry of protons into the mitochondrial matrix independent of ATP synthase. Changes in H(+) leak may affect reactive oxygen species (ROS) production. We measured H(+) leak and ROS production during ischemia-reperfusion and ischemic preconditioning (IPC) and examined how changing mitochondrial respiration affected mΔΨ and ROS production. MATERIALS AND METHODS: Isolated rat hearts (n = 6/group) were subjected to either control-IR or IPC. Rate pressure product (RPP) was measured. Mitochondria were isolated at end reperfusion. Respiration was measured by polarography and titrated with increasing concentrations of malonate (0.5-2 mM). mΔΨ was measured using a tetraphenylphosphonium electrode. H(+) leak is the respiratory rate required to maintain membrane potential at -150 mV in the presence of oligomycin-A. Mitochondrial complex III ROS production was measured by fluorometry using Amplex-red. RESULTS: IPC improved recovery of RPP at end reperfusion (63% ± 4% versus 21% ± 2% in control-IR, P < 0.05). Ischemia-reperfusion caused increased H(+) leak (94 ± 12 versus 31 ± 1 nmol O/mg protein/min in non-ischemic control, P < 0.05). IPC attenuates these increases (55 ± 9 nmol O/mg protein/min, P < 0.05 versus control-IR). IPC reduced mitochondrial ROS production compared with control-IR (31 ± 2 versus 40 ± 3 nmol/mg protein/min, P < 0.05). As mitochondrial respiration decreased, mΔΨ and mitochondrial ROS production also decreased. ROS production remained lower in IPC than in control-IR for all mΔΨ and respiration rates. CONCLUSIONS: Increasing H(+) leak is not associated with decreased ROS production. IPC decreases both the magnitude of H(+) leak and ROS production after ischemia-reperfusion.

Differential effects of non-steroidal anti-inflammatory drugs on mitochondrial dysfunction during oxidative stress.

We investigated the effects of several non-steroidal anti-inflammatory drugs on swelling related properties of mitochondria, with an emphasis on compounds that are marketed and utilized topically in the eye (nepafenac, ketorolac, diclofenac, bromfenac), and compared these to the effects of amfenac (a metabolite of nepafenac) and to celecoxib (active principle of Celebrex). With the exception of the last compound, none of the drugs promote swelling of normal mitochondria that are well energized by succinate oxidation. However, swelling is seen when the mitochondria are under an oxidative stress due to the presence of t-butylhydroperoxide. When used at 200 microM the order of potency is celecoxib > bromfenac > diclofenac > ketorolac > amfenac > nepafenac approximately equal to 0. Again with the exception of celecoxib, this swelling is not seen when mitochondria are depleted of endogenous Ca(2+) and is accelerated when exogenous Ca(2+) is provided. Sr(2+) does not substitute for exogenous Ca(2+) and prevents swelling in the presence of endogenous Ca(2+) only. The same is true for ruthenium red (inhibitor of the Ca(2+) uniporter), for cyclosporin A (inhibitor of the mitochondrial permeability transition), and for a 3.4 kDa polyethylene glycol (polymer that cancels the force which drives swelling following the permeability transition). It is concluded that several non-steroidal anti-inflammatory drugs promote the mitochondrial permeability transition under conditions of oxidative stress and in a Ca(2+) dependent fashion, whereas celecoxib functions by another mechanism. Potency of those compounds that promote the transition varies widely with bromfenac being the most potent and nepafenac having almost no effect. The mitochondrial dysfunction which is caused by the transition may underlie side effects that are produced by some of these compounds.

Ca2+-induced permeability transition can be observed even in yeast mitochondria under optimized experimental conditions.

Yeast mitochondria have generally been believed not to undergo the permeability transition (PT) by the accumulation of Ca(2+) within the mitochondrial matrix, unlike mammalian mitochondria. However, the reason why the yeast PT is not induced by Ca(2+) has remained obscure. In this study, we examined in detail the effects of Ca(2+) on yeast mitochondria under various conditions. As a result, we discovered that the PT could be induced even in yeast mitochondria by externally added Ca(2+) under optimized experimental conditions. The 2 essential parameters for proper observation of the PT-inducing effects of Ca(2+) were the concentrations of the respiratory substrate and that of inorganic phosphate (Pi) in the incubation medium. The yeast mitochondrial PT induced by Ca(2+) was found to be insensitive to cyclosporin A and suppressed in the presence of a high concentration of Pi. Furthermore, when the PT was induced in yeast mitochondria by Ca(2+), the release of cytochrome c from mitochondria was also observed.

Mitochondrial complex II in the post-ischemic heart: oxidative injury and the role of protein S-glutathionylation.

Mitochondrial superoxide (O2.) is an important mediator of ischemia/reperfusion (I/R) injury. The O2. generated in mitochondria also acts as a redox signal triggering cellular apoptosis. The enzyme succinate ubiquinone reductase (SQR or complex II) is one of the major mitochondrial components hosting regulatory thiols. Here the intrinsic protein S-glutathionylation (PrSSG) at the 70-kDa FAD-binding subunit of SQR was detected in rat heart and in isolated SQR using an anti-GSH monoclonal antibody. When rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion, the electron transfer activity (ETA) of SQR in post-ischemic myocardium was significantly decreased by 41.5 +/- 2.9%. The PrSSGs of SQR-70 kDa were partially or completely eliminated in post-ischemic myocardium obtained from in vivo regional I/R hearts or isolated global I/R hearts, respectively. These results were further confirmed by using isolated succinate cytochrome c reductase (complex II + complex III). In the presence of succinate, O2. was generated and oxidized the SQR portion of SCR, leading to a 60-70% decrease in its ETA. The gel band of the S-glutathionylated SQR 70-kDa polypeptide was cut out and digested with trypsin, and the digests were subjected to liquid chromatography/tandem mass spectrometry analysis. One cysteine residue, Cys(90), was involved in S-glutathionylation. These results indicate that the glutathione-binding domain, (77)AAFGLSEAGFNTACVTK(93) (where underline indicates Cys(90)), is susceptible to redox change induced by oxidative stress. Furthermore, in vitro S-glutathionylation of purified SQR resulted in enhanced SQR-derived electron transfer efficiency and decreased formation of the 70-kDa-derived protein thiyl radical induced by O2. . Thus, the decreasing S-glutathionylation and ETA in mitochondrial complex II are marked during myocardial ischemia/reperfusion. This redox-triggered impairment of complex II occurs in the post-ischemic heart and should be useful to identify disease pathogenesis related to reactive oxygen species-induced mitochondrial dysfunction.

Monensin improves the effectiveness of meso-dimercaptosuccinate when used to treat lead intoxication in rats.

Among divalent cations, the ionophore monensin shows high activity and selectivity for the transport of lead ions (Pb2+) across phospholipid membranes. When coadministered to rats that were receiving meso-dimercaptosuccinate for treatment of Pb intoxication, monensin significantly increased the amount of Pb removed from femur, brain, and heart. It showed a tendency to increase Pb removal from liver and kidney but had no effect of this type in skeletal muscle. Tissue levels of several physiologic (calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, zinc) and nonphysiologic (arsenic, cadmium, chromium, nickel, strontium) elements were also determined after the application of these compounds. Among the physiologic elements, a number of significant changes were seen, including both rising and falling values. The size of these changes was typically around 20% compared with control values, with the largest examples seen in femur. These changes often tended to reverse those of similar size that had occurred during Pb administration. Among the nonphysiologic elements, which were present in trace amounts, the changes were smaller in number but larger in size. None of these changes appears likely to be significant in terms of toxicity, and there were no signs of overt toxicity under any of the conditions employed. Monensin may act by cotransporting Pb2+ and OH- ions out of cells, in exchange for external sodium ions. The net effect would be to shuttle intracellular Pb2+ to extracellular dimercaptosuccinic acid thereby enhancing its effectiveness. Thus, monensin may be useful for the treatment of Pb intoxication when applied in combination with hydrophilic Pb2+ chelators.

Selective transport of Pb2+ and Cd2+ across a phospholipid bilayer by a cyclohexanemonocarboxylic acid-capped 15-crown-5 ether.

A cyclohexanemonocarboxylic acid-capped 15-crown-5 ether was synthesized and found to be effective as an ionophore for Pb2+ and Cd2+, transporting them across a phospholipid bilayer membrane. Transport studies were carried out using 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine (POPC) vesicles containing the chelating indicator 2-([2-bis(carboxymethyl)amino-5-methylphenoxy]methyl)-6-methoxy-8-bis(carboxymethyl)aminoquinoline (Quin-2). Data obtained at pH 7.0 using this system, show that the synthetic ionophore transports divalent cations with the selectivity sequence Pb2+ > Cd2+ >> Zn2+ > Mn2+ > Co2+ > Ni2+ > Ca2+ > Sr2+. Selectivity factors, based on the ratio of individual initial cation transport rates, are 280 (Pb2+/Ca2+), 62 (Pb2+/Zn2+), 68 (Cd2+/Ca2+), and 16 (Cd2+/Zn2+). Plots of log initial rate versus logM(n+) or log ionophore concentration suggest that Pb2+ and Cd2+ are transported primarily as a 1:1 cation-ionophore complex, but that complexes with other stoichiometries may also be present. The ionophore transports Pb2+ and Cd2+ by a predominantly electrogenic mechanism, based upon an enhanced rate of transport that is produced by agents which dissipate transmembrane potentials. The rate of Pb2+ transport shows a biphasic pH dependence with the maximum occurring at pH approximately 6.5. The high selectivity for Pb2+ and Cd2+ displayed by the cyclohexanecarboxylic acid-capped 15-crown-5 ether suggests potential applications of this ionophore for the treatment of Pb and Cd intoxication, and removal of these heavy metals from wastewater.

Release of Ca2+ and Mg2+ from yeast mitochondria is stimulated by increased ionic strength.

BACKGROUND: Divalent cations are required for many essential functions of mitochondrial metabolism. Yet the transporters that mediate the flux of these molecules into and out of the mitochondrion remain largely unknown. Previous studies in yeast have led to the molecular identification of a component of the major mitochondrial electrophoretic Mg2+ uptake system in this organism as well as a functional mammalian homolog. Other yeast mitochondrial studies have led to the characterization of an equilibrative fatty acid-stimulated Ca2+ transport activity. To gain a deeper understanding of the regulation of mitochondrial divalent cation levels we further characterized the efflux of Ca2+ and Mg2+ from yeast mitochondria. RESULTS: When isolated mitochondria from the yeast Saccharomyces cerevisiae were suspended in a salt-based suspension medium, Ca2+ and Mg2+ were released from the matrix space. Release did not spontaneously occur in a non-ionic mannitol media. When energized mitochondria were suspended in a mannitol medium in the presence of Ca2+ they were able to accumulate Ca2+ by the addition of the electrogenic Ca2+ ionophore ETH-129. However, in a KCl or choline Cl medium under the same conditions, they were unable to retain the Ca2+ that was taken up due to the activation of the Ca2+ efflux pathway, although a substantial membrane potential driving Ca2+ uptake was maintained. This Ca2+ efflux was independent of fatty acids, which have previously been shown to activate Ca2+ transport. Endogenous mitochondrial Mg2+ was also released when mitochondria were suspended in an ionic medium, but was retained in mitochondria upon fatty acid addition. When suspended in a mannitol medium, metal chelators released mitochondrial Mg2+, supporting the existence of an external divalent cation-binding site regulating release. Matrix space Mg2+ was also slowly released from mitochondria by the addition of Ca2+, respiratory substrates, increasing pH, or the nucleotides ATP, ADP, GTP, and ATP-gamma-S. CONCLUSION: In isolated yeast mitochondria Ca2+ and Mg2+ release was activated by increased ionic strength. Free nucleotides, metal ion chelators, and increased pH also stimulated release. In yeast cells this release is likely an important mechanism in the regulation of mitochondrial matrix space divalent cation concentrations.

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.

Loss of NAD(H) from swollen yeast mitochondria.

BACKGROUND: The mitochondrial electron transport chain oxidizes matrix space NADH as part of the process of oxidative phosphorylation. Mitochondria contain shuttles for the transport of cytoplasmic NADH reducing equivalents into the mitochondrial matrix. Therefore for a long time it was believed that NAD(H) itself was not transported into mitochondria. However evidence has been obtained for the transport of NAD(H) into and out of plant and mammalian mitochondria. Since Saccharomyces cerevisiae mitochondria can directly oxidize cytoplasmic NADH, it remained questionable if mitochondrial NAD(H) transport occurs in this organism. RESULTS: NAD(H) was lost more extensively from the matrix space of swollen than normal, condensed isolated yeast mitochondria from Saccharomyces cerevisiae. The loss of NAD(H) in swollen organelles caused a greatly decreased respiratory rate when ethanol or other matrix space NAD-linked substrates were oxidized. Adding NAD back to the medium, even in the presence of a membrane-impermeant NADH dehydrogenase inhibitor, restored the respiratory rate of swollen mitochondria oxidizing ethanol, suggesting that NAD is transported into the matrix space. NAD addition did not restore the decreased respiratory rate of swollen mitochondria oxidizing the combination of malate, glutamate, and pyruvate. Therefore the loss of matrix space metabolites is not entirely specific for NAD(H). However, during NAD(H) loss the mitochondrial levels of most other nucleotides were maintained. Either hypotonic swelling or colloid-osmotic swelling due to opening of the yeast mitochondrial unspecific channel (YMUC) in a mannitol medium resulted in decreased NAD-linked respiration. However, the loss of NAD(H) from the matrix space was not mediated by the YMUC, because YMUC inhibitors did not prevent decreased NAD-linked respiration during swelling and YMUC opening without swelling did not cause decreased NAD-linked respiration. CONCLUSION: Loss of endogenous NAD(H) from isolated yeast mitochondria is greatly stimulated by matrix space expansion. NAD(H) loss greatly limits NAD-linked respiration in swollen mitochondria without decreasing the NAD-linked respiratory rate in normal, condensed organelles. NAD addition can totally restore the decreased respiration in swollen mitochondria. In live yeast cells mitochondrial swelling has been observed prior to mitochondrial degradation and cell death. Therefore mitochondrial swelling may stimulate NAD(H) transport to regulate metabolism during these conditions.