Influence of growth hormone and thyroxine on thermotropic effects of respiration and 1-anilino-8-naphthalene sulfonate fluorescence and on lipid composition of cardiac membranes

The effects of hypophysectomy and subsequent administration of growth hormone and/or L-thyroxine on thermotropic properties of State 3 respiration (ADP-induced), cholesterol, phospholipid and fatty acid composition of phospholipid fraction were examined in myocardial mitochondria of rats. Temperature-dependence of 1-anilino-8-naphthalene sulfonate fluorescence was determined in vesicles prepared from lipids of heart mitochondria. Transition temperature obtained from the Arrhenius plots of respiration occurred at 21 and 24°C for heart mitochondria of normal and hypophysectomized rats, respectively. Most notably, after hypophysectomy the rate of respiration was lower below 24°C, but was progressively higher above that temperature when compared to normal rats. The energy of activation was 148 and 36% larger below and above the transition temperature, respectively. Growth hormone restored almost completely the energy of activation and respiratory rates to normal levels. Administration of L-thyroxine, with or without growth hormone, did not significantly change the rate of respiration but decreased the transition temperature to 17.7–17.0°C. Lipid and phospholipid content, as well as percent distribution of phospholipids and their fatty acid composition were not statistically different among the different groups of rats. Only cholesterol content was increased after hypophysectomy. Administration of growth hormone and thyroxine did not significantly change the total unsaturation index of fatty acids, but growth hormone increased the content of arachidonic acid (20 : 4) by 70% but decreased the docosahexaenoic acid (22 : 6) three times which may have a beneficial effect on mitochondrial membranes. These and other results suggest that hormones exert different effects on subcellular organelles in different tissues, like heart and liver.     

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.     

Effect of catecholamine depletion on oxidative energy metabolism in rat liver, brain and heart mitochondria; use of reserpine

Regulation of mitochondrial functions in vivo by catecholamines was examined indirectly by depleting the catecholamines stores by reserpine treatments of the experimental animals. Reserpine treatment resulted in decreased respiratory activity in liver and brain mitochondria with the two NAD+-linked substrates: glutamate and pyruvate + malate with succinate ATP synthesis rate decreased in liver mitochondria only. With ascorbate + TMPD system, the ADP/O ratio and ADP phosphorylation rate decreased in brain mitochondria. For the heart mitochondria, state 3 respiration rates decreased for all substrates. In the liver mitochondria basal ATPase activity decreased by 51%, but in the presence of Mg2+ and/or DNP increased significantly. In the brain and heart mitochondria ATPase activities were unchanged. The energy of activation in high temperature range increased liver mitochondrial ATPase while in brain mitochondria reserpine treatment resulted in abolishment in phase transition. Total phospholipid (TPL) content of the brain mitochondria increased by 22%. For the heart mitochondria TPL content decreased by 19% and CHL content decreased by 34%. Tissue specific differential effects were observed for the mitochondrial phospholipid composition. Liver mitochondrial membranes were more fluidized in the reserpine-treated group. The epinephrine and norepinephrine contents in the adrenals decreased by 68 and 77% after reserpine treatment.     

Hormones and liver mitochondria: Influence of growth hormone,thyroxine, testosterone,and insulin on thermotropic effects of respiration and fatty acid composition of membranes

The effects of hypophysectomy and subsequent administration of growth hormone, thyroxine, insulin, and testosterone were examined in rat liver for the relationship between the thermotropic effects on State 3 respiration (ADP induced) and fatty acid composition of the phospholipid fraction of intact mitochondria as well as of inner membrane vesicles. The Arrhenius profile for energy-linked (succinate) State 3 respiration of mitochondria from hypophysectomized rats lacked the discontinuity at 23.5 °C seen with mitochondria from normal rats. After injections of the hormones the discontinuity representing the transition temperature from gel to liquid crystalline state of lipids occurred at different temperatures: 18.5 °C for growth hormone, 26.0 °C for thyroxine, 19.5 °C for growth hormone + thyroxine, 27.6 °C for insulin, and 25.3 °C for testosterone. The energy of activation between 37.5 and 23.5 °C was 1.9 times greater for hypophysectomy than for controls. Growth hormone was the most effective in restoring the energy of activation to normal, above as well as below transition temperature. The effect of thyroxine appears to be due to a larger stimulation of the State 4 respiration than that of growth hormone, insulin, or testosterone, especially at higher temperatures. Phospholipids extracted from intact mitochondria or inner membrane vesicles of hypophysectomized rats contained less arachidonic acid (20:4) and more linoleic acid (18:2) than those of normal rats. In addition, the contents of some of the minor fatty acids were also changed. Calculated unsaturation index showed an 18.8 and 14.9% depletion in unsaturation in whole mitochondria and inner membranes, respectively. Among the different hormones used to treat the hypophysectomized rats, growth hormone was the most effective in restoring the transition temperature and fatty acid composition to normal levels and increasing the gain in body weight. Although the other hormones increased total unsaturation index to some extent, some of the individual fatty acids were affected differently. Good correlation exists between the unsaturation index of mitochondrial fatty acids and transition temperature of State 3 respiration. These results strongly suggest a role for the hormones, particularly growth hormone, in the control of mitochondrial membrane fluidity of hypophysectomized rat liver, through fatty acid composition of phospholipids.     

Cardiolipins and biomembrane function.

Evidence is discussed for roles of cardiolipins in oxidative phosphorylation mechanisms that regulate State 4 respiration by returning ejected protons across and over bacterial and mitochondrial membrane phospholipids, and that regulate State 3 respiration through the relative contributions of proteins that transport protons, electrons and/or metabolites. The barrier properties of phospholipid bilayers support and regulate the slow proton leak that is the basis for State 4 respiration. Proton permeability is in the range 10(-3)-10(-4) cm s-1 in mitochondria and in protein-free membranes formed from extracted mitochondrial phospholipids or from stable synthetic phosphatidylcholines or phosphatidylethanolamines. The roles of cardiolipins in proton conductance in model phospholipid membrane systems need to be assessed in view of new findings by Hübner et al. [313]: saturated cardiolipins form bilayers whilst natural highly unsaturated cardiolipins form nonlamellar phases. Mitochondrial cardiolipins apparently participate in bilayers formed by phosphatidylcholines and phosphatidylethanolamines. It is not yet clear if cardiolipins themselves conduct protons back across the membrane according to their degree of fatty acyl saturation, and/or modulate proton conductance by phosphatidylcholines and phosphatidylethanolamines. Mitochondrial cardiolipins, especially those with high 18:2 acyl contents, strongly bind many carrier and enzyme proteins that are involved in oxidative phosphorylation, some of which contribute to regulation of State 3 respiration. The role of cardiolipins in biomembrane protein function has been examined by measuring retained phospholipids and phospholipid binding in purified proteins, and by reconstituting delipidated proteins. The reconstitution criterion for the significance of cardiolipin-protein interactions has been catalytical activity; proton-pumping and multiprotein interactions have yet to be correlated. Some proteins, e.g., cytochrome c oxidase are catalytically active when dimyristoylphosphatidylcholine replaces retained cardiolipins. Cardiolipin-protein interactions orient membrane proteins, matrix proteins, and on the outerface receptors, enzymes, and some leader peptides for import; activate enzymes or keep them inactive unless the inner membrane is disrupted; and modulate formation of nonbilayer HII-phases. The capacity of the proton-exchanging uncoupling protein to accelerate thermogenic respiration in brown adipose tissue mitochondria of cold-adapted animals is not apparently affected by the increased cardiolipin unsaturation; this protein seems to take over the protonophoric role of cardiolipins in other mitochondria. Many in vivo influences that affect proton leakage and carrier rates selectively alter cardiolipins in amount per mitochondrial phospholipids, in fatty acyl composition and perhaps in sidedness; other mitochondrial membrane phospholipids respond less or not at all.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diversity of the human erythrocyte membrane sialic acids in relation with blood groups

The aim of the present study was to detect defective structural properties in bilayers of mitochondrial phospholipids after oxidative stress of isolated mitochondria in vitro, reportedly during respiration state IV. The structural behaviour of extracted phospholipids was studied by electron paramagnetic resonance (EPR) spectrometry in oriented phospholipid bilayers spin-labelled with 5-doxyl-lecithin, by detecting of the degree of EPR spectral anisotropy loss, indicative of the phospholipid bilayer packing order. Bilayers of phospholipids from untreated mitochondria showed the highest spectral anisotropy, hence highly ordered structure, while chemically oxidised phospholipid yielded almost completely disordered supported phospholipid bilayers. Samples from mitochondria after respiration state IV showed bilayer disorder increasing with oxidation time, while inclusion of the antioxidant resveratrol in the respiration medium almost completely prevented bilayer disordering. On the other hand, β-n-doxylstearoyl-lecithin spin-labelled mitochondria showed unchanged order parameter S at C positions 5, 12 and 16 after respiration state IV, confirming the insensitivity of this parameter to phospholipid oxidative stress. It is concluded that reactive oxygen species attack to the membrane affects lipid packing order more than fluidity, and that EPR anisotropy loss reveals oxidative damage to the bilayer better than the order parameter.     

Structure and function of mitochondria in hepatopancreas cells from metabolically depressed snails

Mitochondria in cells isolated from the hepatopancreas of aestivating land snails (Helix aspersa) consume oxygen at 30% of the active control rate. The aim of this study was to investigate whether the lower respiration rate is caused by a decrease in the density of mitochondria or by intrinsic changes in the mitochondria. Mitochondria occupied 2% of cellular volume, and the mitochondrial inner membrane surface density was 17 microm(-1), in cells from active snails. These values were not different in cells from aestivating snails. The mitochondrial protein and mitochondrial phospholipid contents of cells were also similar. There was little difference in the phospholipid fatty acyl composition of mitochondria isolated from metabolically depressed or active snails, except for arachidonic acid, which was 18% higher in mitochondria from aestivating snails. However, the activities of citrate synthase and cytochrome c oxidase in mitochondria isolated from aestivating snails were 68% and 63% of control, respectively. Thus the lower mitochondrial respiration rate in hepatopancreas cells from aestivating snails was not caused by differences in mitochondrial volume or surface density but was associated with intrinsic changes in the mitochondria.     

Inhibition of brain mitochondrial respiration by dopamine: involvement of H(2)O(2) and hydroxyl radicals but not glutathione-protein-mixed disulfides

Examination of the downstream mediators responsible for inhibition of mitochondrial respiration by dopamine (DA) was investigated. Consistent with findings reported by others, exposure of rat brain mitochondria to 0.5 mm DA for 15 min at 30 degrees C inhibited pyruvate/glutamate/malate-supported state-3 respiration by 20%. Inhibition was prevented in the presence of pargyline and clorgyline demonstrating that mitochondrial inhibition arose from products formed following MAO metabolism and could include hydrogen peroxide (H(2) O(2) ), hydroxyl radical, oxidized glutathione (GSSG) or glutathione-protein mixed disulfides (PrSSG). As with DA, direct incubation of intact mitochondria with H(2) O(2) (100 microm) significantly inhibited state-3 respiration. In contrast, incubation with GSSG (1 mm) had no effect on O(2) consumption. Exposure of mitochondria to 1 mm GSSG resulted in a 3.3-fold increase in PrSSG formation compared with 1.4- and 1.5-fold increases in the presence of 100 microm H(2) O(2) or 0.5 mm DA, respectively, suggesting a dissociation between PrSSG formation and effects on respiration. The lack of inhibition of respiration by GSSG could not be accounted for by inadequate delivery of GSSG into mitochondria as increases in PrSSG levels in both membrane-bound (2-fold) and intramatrix (3.5-fold) protein compartments were observed. Furthermore, GSSG was without effect on electron transport chain activities in freeze-thawed brain mitochondria or in pig heart electron transport particles (ETP). In contrast, H(2) O(2) showed differential effects on inhibition of respiration supported by different substrates with a sensitivity of succinate > pyruvate/malate > glutamate/malate. NADH oxidase and succinate oxidase activities in freeze-thawed mitochondria were inhibited with IC(50) approximately 2-3-fold higher than in intact mitochondria. ETPs, however, were relatively insensitive to H(2) O(2). Co-administration of desferrioxamine with H(2) O(2) had no effect on complex I-associated inhibition in intact mitochondria, but attenuated inhibition of rotenone-sensitive NADH oxidase activity by 70% in freeze-thawed mitochondria. The results show that DA-associated inhibition of respiration is dependent on MAO and that H(2) O(2) and its downstream hydroxyl radical rather than increased GSSG and subsequent PrSSG formation mediate the effects.     

Effects of Fluoride on Mitochondrial Activity in Higher Plants

The effects of fluoride on respiration of plant tissue and mitochondria were investigated. Fumigation of young soybean plants (Glycine max Merr. cv. Hawkeye) with 9–12 μg × m^?3 HF caused a stimulation of respiration at about 2 days of treatment followed by inhibition 2 days later. Mitochondria isolated from the stimulated tissue had higher respiration rates, greater ATPase activity, and lower P/O ratios, while in mitochondria from inhibited tissue, all three were reduced. Treatment of etiolated soybean hypocotyl sections in Hoagland's solution containing KF for 3 to 10 h only resulted in inhibition of respiration. Mitochondria isolated from this tissue elicited increased respiration rates with malate as substrate and inhibited respiration with succinate. With both substrates respiratory control and ADP/O ratios were decreased. Direct treatment of mitochondria from the etiolated soybean hypocotyl tissue with fluoride resulted in inhibition of state 3 respiration and lower ADP/O ratios with the substrates succinate, malate, and NADH. Fluoride was also found to increase the amount of osmotically induced swelling and cause a more rapid leakage of protein with mitochondria isolated from etiolated corn shoots (Zea mays L. cv. Golden Cross Bantam). The results are discussed with respect to possible effects of fluoride on mitochondrial membranes.     

Changes in phospholipid composition of mitochondria after freezing-warming in the presence of Na2S204

The phospholipid composition of mitochondria membranes subjected to rapid freezing (300-400 degrees C per 1 min) to -196 degrees C and subsequent slow warning (at 20 degrees C) was determined by the method of chromatography in the thin layer of silicagel. Under such conditions of freezing and warming a significant decrease in lecithin and ethanolamine phosphatide content is observed in the mitochondria membranes. When freezing the suspension of mitochondria in the medium containing Na2S2O4 in a concentration of 0.05 M only the lipid component changes slightly.     

Comparison of membrane organization in mitochondria from yeast and rat liver by nuclear magnetic resonance spectroscopy

Summary Proton magnetic resonance (PMR) and carbon-13 magnetic resonance (CMR) spectra of intact, unsonicated yeast and rat liver motochondria show differences which may be correlated with the composition of the membranes. High resolution PMR and CMR signals in intact yeast mitochondria have been assigned to regions of fluid lipid-lipid interaction on the basis of spectra of extracted lipid and protein, and the temperature dependence of NMR signals from the intact membrane. PMR spectra suggest that about 20% of total yeast phospholipid is in regions where both intramolecular fatty acid chain mobility and lateral diffusion of entire phospholipid molecules are possible. No such regions appear to exist in rat liver mitochondria. For both yeast and rat liver mitochondria, comparison of PMR and CMR spectra suggests that about 50% of phospholipid appears to be in regions where intramolecular fatty acid chain motion is considerable, but lateral diffusion is restricted. The remaining phospholipid appears to have little inter- or intramolecular mobility. Since NMR observation of lipid extracts from membranes indicates that phospholipid-sterol interactions do not account for the spectra of intact mitochondria, these effects are interpreted in terms of extensive lipid-protein interactions.     

Incorporation and subcellular distribution of an unnatural phospholipid base-analog, N-isopropyl-ethanolamine, in rat liver.

An unnatural phospholipid, phosphatidyl-N-isopropylethanolamine, was isolated from rat liver after intraperitoneal injections of N-isopropylethanol-amine; it was identified on the basis of enzymic, chemical, and chromatographic analyses. Although this phospholipid was formed at the expense of phosphatidylcholine and phosphatidylethanolamine, its fatty acid composition did not resemble either of these lipids. Microsomes, mitochondria, and plasma membranes contained significant amounts (up to 9%) of this unusual phospholipid. Radioisotope incorporation experiments suggest that the N-isopropylethanol-amine containing phospholipid is rapidly equilibrated between microsomes and mitochondria and more slowly with surface membranes.     

Ca2+ transport mediated by a synthetic neutral Ca2+ -ionophore in biological membranes.

The effect of a synthetic neutral ligand on the Ca2+ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca2+ -ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca2+ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) are present, presumably to facilitate the diffusion of the charged Ca2+ -ionophore complex across the hydrophobic core of the membrane. In mitochondria, the neutral ionophore promotes the active transport of Ca2+ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red.     

Brain mitochondrial nitric oxide synthase: in vitro and in vivo inhibition by chlorpromazine

Mouse brain mitochondria have a nitric oxide synthase (mtNOS) of 147 kDa that reacts with anti-nNOS antibodies and that shows an enzymatic activity of 0.31-0.48 nmol NO/min mg protein. Addition of chlorpromazine to brain submitochondrial membranes inhibited mtNOS activity (IC50 = 2.0 +/- 0.1 microM). Brain mitochondria isolated from chlorpromazine-treated mice (10 mg/kg, i.p.) show a marked (48%) inhibition of mtNOS activity and a markedly increased state 3 respiration (40 and 29% with malate-glutamate and succinate as substrates, respectively). Respiration of mitochondria isolated from control mice was 16% decreased by arginine and 56% increased by NNA (Nomega-nitro-L-arginine) indicating a regulatory activity of mtNOS and NO on mitochondrial respiration. Similarly, mitochondrial H2O2 production was 55% decreased by NNA. The effect of NNA on mitochondrial respiration and H2O2 production was significantly lower in chlorpromazine-added mitochondria and absent in mitochondria isolated from chlorpromazine-treated mice. Results indicate that chlorpromazine inhibits brain mtNOS activity in vitro and can exert the same action in vivo.     

Lipid-protein interactions in mitochondria. The effect of protein interaction on susceptibility of phospholipids to phospholipase A2 and C hydrolysis.

Phospholipase A₂ (Naja naja) and phospholipase C (from either Clostridium welchii or Bacillus cereus) have been tested on phospholipid dispersions and natural or reconstituted membranes; notwithstanding the different substrate specificities, the different enzymes gave comparable behaviors, suggesting that the results were the expression of sterical features in the lipid bilayers, i.e., availability of the phospholipids to enzymatic attack. The hydrolysis of phospholipids (Asolectin) in sonic protein-free vesicles is hindered by ionic interaction with basic proteins (cytochrome c or lysozyme). On the other hand binding of Asolectin to lipid-depleted mitochondria to obtain reconstituted mitochondria does not prevent phospholipase action on the phospholipids; similarly, phospholipids are hydrolyzed at maximal rates in natural membranes (mitochondria or submitochondrial particles). Surprisingly, ionic interaction of RM or natural membranes with basic proteins does not prevent phospholipase hydrolysis of the membrane phospholipids. The interpretation of this phenomenon may be related to the heterogeneity of phospholipid distribution in protein-containing membranes.     

Anti-apoptotic Bcl-2 Family Proteins Disassemble Ceramide Channels

Early in mitochondria-mediated apoptosis, the mitochondrial outer membrane becomes permeable to proteins that, when released into the cytosol, initiate the execution phase of apoptosis. Proteins in the Bcl-2 family regulate this permeabilization, but the molecular composition of the mitochondrial outer membrane pore is under debate. We reported previously that at physiologically relevant levels, ceramides form stable channels in mitochondrial outer membranes capable of passing the largest proteins known to exit mitochondria during apoptosis (Siskind, L. J., Kolesnick, R. N., and Colombini, M. (2006) Mitochondrion 6, 118-125). Here we show that Bcl-2 proteins are not required for ceramide to form protein-permeable channels in mitochondrial outer membranes. However, both recombinant human Bcl-x(L) and CED-9, the Caenorhabditis elegans Bcl-2 homologue, disassemble ceramide channels in the mitochondrial outer membranes of isolated mitochondria from rat liver and yeast. Importantly, Bcl-x L and CED-9 disassemble ceramide channels in the defined system of solvent-free planar phospholipid membranes. Thus, ceramide channel disassembly likely results from direct interaction with these anti-apoptotic proteins. Mutants of Bcl-x L act on ceramide channels as expected from their ability to be anti-apoptotic. Thus, ceramide channels may be one mechanism for releasing pro-apoptotic proteins from mitochondria during the induction phase of apoptosis.     

大鼠心肌线粒体内、外膜磷脂动态结构的研究

我们以DPH为荧光探针.用毫微秒荧光分光光度计测定了大鼠心肌线粒体及线粒体内、外膜的动态微细结构;用HPLC分析了磷脂组成.实验结果提示.完整线粒体膜流动性主要反映了线粒体外膜的运动状态.线粒体内膜微粘度及磷脂分子摇动角大于外膜,扩散速率小于外膜.除去了蛋白质的线粒体内、外膜磷脂脂质体膜流动性无明显差异.提示线粒体内膜的高微粘度与膜中所含有的多量蛋白有关.     

Newly made phosphatidylserine and phosphatidylethanolamine are preferentially translocated between rat liver mitochondria and endoplasmic reticulum

The translocation of: (i) phosphatidylserine (PtdSer) from its site of synthesis on microsomal membranes to its site decarboxylation in mitochondrial membranes and (ii) phosphatidylethanolamine (PtdEtn) from the mitochondria to its site of methylation to phosphatidylcholine on microsomal membranes has been reconstituted in cell-free systems consisting of rat liver mitochondria and microsomes. Two types of systems have been reconstituted. In one, the translocation of newly made PtdSer or PtdEtn was examined by incubation of microsomes and mitochondria with [3-3H]serine. In the other, membranes were prelabeled with radioactive PtdSer or PtdEtn, and the transfer of these two lipids between mitochondria and microsomes was monitored. For the transfer of both PtdSer from microsomes to mitochondria and PtdEtn from mitochondria to microsomes, newly made phospholipids were translocated much more readily than pre-existing phospholipids. The data suggest that with respect to their translocation between these two organelles, the pools of newly synthesized PtdSer and PtdEtn were distinct from the pools of "older" phospholipids pre-existing in the membranes. Transfer of neither phospholipid in vitro depended on the presence of cytosolic proteins (i.e. soluble phospholipid transfer proteins) or on the hydrolysis of ATP, although there was some stimulation of PtdSer transfer by ATP and several other nucleoside mono-, di-, and triphosphates. The data are consistent with a collision-based mechanism in which the endoplasmic reticulum and mitochondria come into contact with one another, thereby effecting the transfer of phospholipids. The proposal that there is contact between the endoplasmic reticulum and mitochondria is supported by the recent isolation of a membrane fraction having many, but not all, of the properties of the endoplasmic reticulum, but which was isolated in association with mitochondria (Vance, J. E. (1990) J. Biol. Chem. 265, 7248-7256).     

Role of charge screening and delocalization for lipophilic cation permeability of model and mitochondrial membranes

The effects of the mitochondria-targeted lipophilic cation dodecyltriphenylphosphonium (C₁₂TPP, the charge is delocalized and screened by bulky hydrophobic residues) and those of lipophilic cations decyltriethylammonium bromide and cetyltrimethylammonium bromide (C₁₀TEA and C₁₆TMA, the charges are localized and screened by less bulky residues) on bilayer planar phospholipid membranes and tightly-coupled mitochondria from the yeast Yarrowia lipolytica have been compared. In planar membranes, C₁₂TPP was found to generate a diffusion potential as if it easily penetrates these membranes. In the presence of palmitate, C₁₂TPP induced H⁺ permeability like plastoquinonyl decyltriphenilphosphonium that facilitates transfer of fatty acid anions (Severin et al., PNAS, 2010, 107, 663–668). C₁₂TPP was shown to stimulate State 4 respiration of mitochondria and caused a mitochondrial membrane depolarization with a half-maximal effect at 6 μM. Besides, C₁₂TPP profoundly potentiated the uncoupling effect of endogenous or added fatty acids. C₁₀TEA and C₁₆TMA inhibited State 4 respiration and decreased the membrane potential, though at much higher concentrations than C₁₂TPP, and they did not promote the uncoupling action of fatty acids. These relationships were modeled by molecular dynamics. They can be explained by different membrane permeabilities for studied cations, which in turn are due to different availabilities of the positive charge in these cations to water dipoles.     

Therapeutic doses of menadione reduce the rotenone-induced inhibition of respiration and membrane potential generation in mitochondria

Menadione restores the rotenone-inhibited respiration of diaphragm muscle pieces in approximately the same degree as the respiration of heart mitochondria, i.e., to 30-40%. The respiration of heart mitochondria induced by 2-5 microM menadione (after its inhibition by rotenone) is partly coupled with ATP synthesis whose rate is much lower than that of oxidation of NAD-dependent substrates. The effects of menadione and mitochondrial energetics inhibitors on lymphocyte respiration and rhodamine 123 fluorescence in individual lymphocytes and their suspensions were compared. Menadione (2--5 microM) increased the rotenone + oligomycin suppressed delta psi m in lymphocytes. At 5-40 microM menadione did not act as an uncoupler and had little effect on the uncoupled lymphocyte respiration. All these effects were observed at menadione concentrations close to therapeutic ones. Vicasol, a water-soluble analog of menadione, exerted a similar effect.